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Sperm guidance in — an unpaved road to the egg

Michael Eisenbach* and Laura C. Giojalas‡ Abstract | Contrary to the prevalent view, there seems to be no competition in the mammalian female genital tract among large numbers of cells that are racing towards the egg. Instead, small numbers of the ejaculated sperm cells enter the , and these few must be guided to make the remaining long, obstructed way to the egg. Here, we review the mechanisms by which mammalian sperm cells are guided to the egg.

Capacitation For fertilization to occur in mammals, ejaculated sperma- — the directed movement of cells along a temperature A ripening process that tozoa must reach the egg, which, following ovulation, gradient. spermatozoa must undergo in has moved from the ovary into the Fallopian tube. Until was discovered as a form of sperm guid- order to penetrate the female’s not too long ago, the common belief was that, in mam- ance in the mid-1960s in marine species9,10, and has egg and fertilize it. mals, following ejaculation into the female genital tract, only been recognized in amphibians and mammals in 7,11 large numbers of spermatozoa ‘race’ towards the egg and the past 15 years . The initial resistance to the notion The release of proteolytic compete to fertilize it. This dogma, as well as conflicting of in mammals was compounded by enzymes from the top part results in the literature, instilled the idea that the guid- the inconsistent conclusions drawn from initial studies of the sperm’s head, known as ance of sperm to the egg was superfluous in mammals. (see REFS 3,11 for reviews). One of the main reasons for the acrosome, which enables sperm penetration through the The ‘competitive-race model’ dismantled when it became the apparent inconsistency was the very low signal-to- egg coat. clear that, in fact, few of the ejaculated spermatozoa (in noise ratio that was obtained in chemotaxis assays with humans, only ~1 of every million spermatozoa) succeed mammalian spermatozoa. This factor, combined with Chemotaxis in entering the Fallopian tubes1–3. Furthermore, the suboptimal experimental conditions and a failure to The movement of cells in the number of spermatozoa that can fertilize the egg is even distinguish between true chemotaxis and other causes direction of a chemoattractant gradient. smaller. Spermatozoa must undergo a process of ripening, of sperm accumulation (reviewed in REF. 3), resulted in known as , and only capacitated spermatozoa conflicting results and ambiguity. Today, we know that Chemoattractant can penetrate the cumulus layer that surrounds the egg, the low signal-to-noise ratio is caused by the fact that, in A factor (a peptide or any bind to the sperm receptor on the egg coat, and undergo mammals (unlike sperm chemotaxis in marine species9), other chemical) that attracts acrosome reaction specific cells by chemotaxis. the that enables sperm penetration only the small fraction of capacitated spermatozoa are through the egg coat and then fusion with the egg (see chemotactically responsive5,12–14. This realization, com- Thermotaxis REF. 4 for a review). bined with the development of assays that allow chemo- The movement of cells that is The percentage of capacitated spermatozoa is low (~10% taxis to be distinguished from other processes7 (BOX 1), directed according to a in humans)4–6 and, therefore, the number of spermatozoa has established that chemotaxis is indeed a mammalian temperature gradient. that can reach and fertilize the egg is small. The chances that sperm-guidance mechanism. such low numbers of spermatozoa will successfully reach The first experiments in mammals showed that human follicular fluid15,16 *Department of Biological the egg by coincidence, without a guidance mechanism, sperm accumulated in the , and that there Chemistry, the Weizmann are very slim. This realization has fuelled an interest in was a remarkable correlation between this in vitro accum- Institute of Science, 76100 the study of in mammals, and here we ulation and egg fertilization16. Subsequent experiments Rehovot, Israel. summarize the state of the art in this young and rapidly confirmed that this accumulation was indeed the con- ‡ Center for Cell and Molecular changing field, comment on new findings and discuss the sequence of chemotaxis17. Sperm chemotaxis was later , Faculty of Sciences, 18 14,19 13 National University of direction in which this research will probably go. also demonstrated in frogs , mice and rabbits . In Córdoba, Avenue Vélez addition, the accumulation of sperm in follicular fluid Sársfield 1601, Mechanisms of mammalian sperm guidance (but without substantiating that it truly reflects chemo- 5000, Córdoba, Argentina. Two active mechanisms of sperm guidance have been taxis) was demonstrated in horses20 and pigs21. So, sperm Correspondence to M.E. chemotaxis e-mail: shown in mammals: , which is the movement chemotaxis seems to be a widespread mechanism that [email protected] of cells up a concentration gradient of chemoattractant guides spermatozoa to the egg, both when fertilization is doi:10.1038/nrm1893 (see REF. 7 for an extensive review), and thermotaxis8 external (as in marine species) or internal.

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5 Box 1 | The measurement of sperm chemotaxis that reach the storage site, only some become capacitated . These few spermatozoa have to negotiate the long (2–6 cm In sperm chemotaxis assays, it is essential to apply well-defined criteria that distinguish in humans1,23 and ~10 cm in rabbits24) obstructed road between chemotaxis and other processes that might cause sperm accumulation. that separates them from the egg at the fertilization site25. There are two main criteria: the directional change of sperm movement towards the In vitro findings indicate that capacitated spermatozoa chemoattractant source — a unique feature of chemotaxis, which, unfortunately, is fulfilled in only the first two assays that are commonly used to measure sperm are guided from the storage site to the egg primarily by a 5,26 8 chemotaxis (see below), and a peak-like dependence (rather than a saturation-curve combination of chemotaxis and thermotaxis , assisted 27 dependence) of the measured response on the chemoattractant concentration. perhaps by oviductal contractions . The latter criterion applies to all chemotaxis assays and it relies on the fact that when a It would seem logical that the capacitated spermatozoa certain chemoattractant concentration saturates its cognate receptor, the cell cannot at the storage site use an ovulation-dependent temperature sense any further increases in the chemoattractant concentration and, as a result, the gradient between this site and the fertilization site24,28,29 as chemotactic response drops. a ‘road sign’ to guide them to the site of fertilization by The assays listed below have been used to determine sperm chemotaxis. In all of them, thermotaxis. Additionally, the finding that, at least in mice, a comparison is made to a no-gradient situation as a control. oviductal fluid contains one or more chemo attractants14 Directionality assays raises the possibility that different chemoattractants might The video-recorded movement tracks of spermatozoa in a chemoattractant gradient be secreted along the to promote sequential, are analysed to determine which tracks involve directional changes towards the source short-range chemotactic processes towards these chemo- of the chemoattractant. The gradient can be two-dimensional (when the source is attractants. As the spermatozoa approach the vicinity point-like)17,35,36,49 or linear (when the source is linear)13. of the fertilization site, they probably sense a chemo- Descending chemoattractant gradient assays attractant gradient that originates at the cumulus cells26 (see Spermatozoa are suspended in a solution containing a chemoattractant and below), which guides them to the egg–cumulus complex. their accumulation at the bottom of a chemoattractant gradient is compared with their However, the arrival at this complex does not guarantee accumulation at the same location when the chemoattractant concentration is 30 constant throughout the measuring unit (a no-gradient control)11,17. Chemotactically entry into the egg itself , because the egg is covered with responsive spermatozoa (unlike chemokinetically responsive spermatozoa that respond a dense matrix that is composed of hundreds of cumulus to the chemoattractant by only increasing their speed of movement) are expected to cells (FIG. 1). It is thought that a chemoattractant gradient accumulate to a lesser extent in the former setup. Trapped spermatozoa would not that originates at the egg is established within the cumulus distinguish between the presence and absence of a gradient. Therefore, in most cases, matrix, which guides the spermatozoa to the egg26. Indeed, this assay allows a distinction between chemotaxis, chemokinesis and trapping. the first few spermatozoa that enter the cumulus are Sperm accumulation in an ascending chemoattractant gradient successful in finding the egg31. The number of spermatozoa that sense an ascending chemoattractant gradient and accumulate near or at its source is counted. Because sperm accumulation can also be Mammalian sperm chemotaxis caused by trapping or changes in swimming speed, this method cannot distinguish Physiology of sperm chemotaxis. The encounter between between chemotaxis and other causes of sperm accumulation11. the gametes of marine species, in which fertilization is Choice assay external, occurs in a turbulent, aqueous environment that Spermatozoa ‘choose’ between a chemoattractant-containing and a chemoattractant- usually contains eggs and spermatozoa from various spe- free well. Such assays can distinguish between chemotaxis and chemokinesis. cies. Therefore, the aim of the long-recognized process However, because a higher sperm concentration near the chemoattractant-containing of sperm chemotaxis in these is suggested to be well can be also caused by trapping, these assays cannot distinguish between not only to recruit as many spermatozoa as possible to chemotaxis and trapping11,17. the eggs, but also to prevent cross-species fertilization. Accordingly, the main physiological characteristics of sperm chemotaxis in marine species include: first, the Follicular fluid In thermotaxis, spermatozoa are directed towards a responsiveness of a large fraction of the sperm population A fluid consisting of sex steroid higher temperature and this mechanism of sperm guid- and, second, in most genera, a high species specificity9,10 hormones, plasma proteins, ance has so far been demonstrated in only two species: (that is, a chemoattractant for one marine species is usu- mucopolysaccharides and humans and rabbits8. Chemotaxis and thermotaxis seem ally not recognized by the spermatozoa of another marine electrolytes that surrounds the ovum in the vesicular ovarian to have a similar function — to guide capacitated, ‘ready- species). By contrast, in mammals, semen is placed follicle (Graafian follicle). to-fertilize’ spermatozoa towards the egg that resides at inside the female genital tract, and sperm competition, the fertilization site. Another potential passive guidance if it exists, is limited to semen from different individuals Oviduct mechanism might be the drag of spermatozoa by muscle of the same species32. It is therefore not surprising that A tube between the ovary and 22 the , through which the contractions in the female genital tract . The existence of species specificity was not detected in experiments that egg is transported from the two or more sperm-guidance mechanisms implies that compared the chemotactic responsiveness of sperma- former to the latter and in sperm guidance is essential for fertilization. tozoa of a few mammalian species to follicular fluids or which fertilization occurs. It egg-conditioned media33. consists of two parts: the Mammalian sperm guidance in the female genital tract. An intriguing question is how large the fraction of isthmus — a narrow part that is closer to the uterus — and the In mammals, the semen is ejaculated either into the vagina chemotactically responsive spermatozoa in mammals is. ampulla — a wider part that is (in primates, ruminants and rabbits) or into the uterus (in On the one hand, several studies concluded that, in human closer to the ovary. rodents, pigs and horses)1. From there, spermatozoa of the and rabbit spermatozoa, only capacitated spermatozoa — former group use a combination of active swimming and which constitute a small fraction of the sperm population Cumulus cells 5,6,13,34 The cells that form dense passive drag through muscle contraction to reach the stor- — are chemotactically responsive . This conclusion layers surrounding a age site in the isthmus of the oviduct (FIG. 1; BOX 2). In vitro was based on the similarity between capacitated and mature egg. experiments imply that, of the few hundreds spermatozoa chemotactically responsive spermatozoa in terms of the

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Thermotaxis Uterus (7–9 cm) Isthmus Chemotaxis (2–3 cm) ~34.7°C ~36.3°C Ampulla Egg (5–8 cm) Oviduct Fertilization Cumulus cell Storage site site

Ovary

Cervix (2–3 cm) Muscle contractions

Acrosome Nucleus Head Mitochondrion

Midpiece Axoneme

Vagina (7–9 cm) Tail (flagellum) Tail Principal piece Figure 1 | The mammalian female genital tract and possible guidance mechanisms. The diagram, which is not drawn to scale, and the dimensions shown are derived from studies in humans1, whereas the temperature values shown are those measured in rabbits24 (there are no published measurements in humans). The dimensions in parentheses indicate the length of the respective organ. The spermatozoa in the vagina use both active swimming and passive drag by female genital-tract muscular contraction to reach the storage site in the oviduct. Once here, a small fraction of spermatozoa undergoes ripening, or capacitation, which enables them to fertilize the egg at the fertilization site. Capacitated spermatozoa are guided from the storage site to the egg by a combination of chemotaxis, thermotaxis and, perhaps, oviductal contractions. An ovulation-dependent temperature gradient between the storage site and the fertilization site provides the thermotactic stimulus. Chemoattractants are present in the oviductal fluid and are also secreted by the egg and the surrounding cumulus cells — providing the chemotactic stimulus that guides the spermatozoa to the egg–cumulus complex. The insert shows a human , which comprises the head (with the acrosome and the nucleus) and the tail or the , consisting of the midpiece (where mitochondria are located around the axoneme) and the principal piece.

size of the fraction in the sperm population, and on the other published chemotaxis assays and compared with the observation that their lifespans are similarly short (see fraction of cells exhibiting Ca2+ elevation in their own below) and that they similarly undergo, and with similar experiments36) awaits clarification. The discrepancy is kinetics, continuous replacement (thereby ensuring the not due to the chemoattractant used, because it was noted continuous availability of capacitated and chemotactically that the chemotactic response of human spermatozoa to responsive spermatozoa in spite of their short lifespan). In bourgeonal was as fractional and small as the response addition, this conclusion was also based on the fact that the to other chemoattractants (M.E., unpublished data). deliberate depletion of capacitated spermatozoa results in The capacitated and chemotactic responsive state is a total loss of chemotaxis and, vice versa, the depletion of neither static nor prolonged. Rather, it is transient and chemotactic spermatozoa results in the depletion of capaci- short (1–4 hours in humans, as measured in vitro), and tated spermatozoa (see REF. 34 for a review). A similar occurs only once in the sperm’s lifetime5,12 (3–5 days in fractional chemotactic responsiveness was observed in humans37,38). Nevertheless, as different spermatozoa mouse spermatozoa14,19. become capacitated at different times, this continuous On the other hand, Spehr et al.35 showed that ~90% replacement of capacitated cells in the sperm population of the motile cells in human spermatozoa swim up the ensures that capacitated spermatozoa are present in the gradient of a chemoattractant known as bourgeonal. female genital tract for extended periods5. This is essential The same group noticed, however, that only about one in species that ovulate periodically (for example, humans), third of the cells responded to bourgeonal with a rise in in whom this extended, continuous replacement probably the intracellular level of Ca2+ — a response thought to ensures the availability of capacitated and chemotactic 36 Axoneme occur in every cell that responds chemotactically . The competent cells throughout the lifespan of spermatozoa 5,6 An axial filament complex at reason for the much higher fraction of responsive cells in in the female genital tract . The extended availability of the centre of the sperm tail. the chemotaxis assays of Spehr et al.35,36 (compared with capacitated, chemotactic spermatozoa seems to be less

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Hydrozoa essential in species with induced ovulation (for example, Non-stimulated Stimulated A class of radially symmetrical rabbits), in which ovulation is induced by mating. In such spermatozoa spermatozoa marine or freshwater species, capacitation seems to be synchronized with ovu- a of the phylum lation6. It therefore seems that the timing of the availability Cnidaria, with one end of the of capacitated, chemotactic spermatozoa is programmed body bearing the mouth and tentacles. This class includes in mammals according to the time at which an ovulated polyps and medusa. egg is available in the female genital tract6. Chemoattractant source Hydromedusa Behavioural mechanisms of chemotaxis. Chemotaxis is A hydrozoan in the medusoid b stage of its life cycle. characterized by directional changes in the movement towards the source of the chemoattractant. Therefore, Ascidian the most direct assays for the measurement of sperm A marine chemotaxis are based on the direction of sperm move- that has a transparent sac- ment7,11. The behaviour of spermatozoa in response to Figure 2 | Two types of sperm response to shaped body with openings chemoattractants. The green and blue lines represent through which water passes; a chemo attractant depends on their variable swimming also known as sea squirt. patterns. In the most well-studied spermatozoa of marine tracks made by swimming spermatozoa. a | Non-stimulated and freshwater species9,39, the swimming patterns and the spermatozoa swim either in almost straight or in curved Chemokinesis changes that occur in response to a chemoattractant can lines. In the presence of a chemoattractant gradient, the The speed enhancement of direction of the swimming movements changes abruptly be classified into two categories (FIG. 2). The first category actively moving cells in towards the chemoattractant source. The drawn tracks are response to a stimulus. includes spermatozoa that swim in almost straight or based on the swimming of human spermatozoa as reported curved lines but, when exposed to a chemoattractant in REFS 17,49. b | Non-stimulated spermatozoa swim in gradient, there is an abrupt change in the direction concentric circles that, when in a chemoattractant of swimming towards the chemoattractant source. gradient, change into loops towards the chemoattractant Spermatozoa of hydrozoa (for example, Campanularia source. The drawn tracks are based on the swimming of flexuosa and Gonionemus vertens) belong to this cate- ascidian and sea-urchin spermatozoa as reported in REF. 40 gory9. The second category includes spermatozoa that and REFS 43,66, respectively. swim in circles but, when exposed to a chemoattractant gradient, the circles become biased towards the chemo- attractant source, resulting in a spiral path towards the the behavioural response of these spermatozoa in a gradient. The spermatozoa of hydromedusa, ascidian, chemo attractant gradient. What is known is that when and belong to this category9,40,41. human spermatozoa are swimming in an ascending A detailed model of the intracellular molecular events chemoattractant gradient, they beat their flagella that mediate the formation of this spiral path has recently symmetrically and reach the chemoattractant source been proposed by Böhmer et al.41 Unique features of this by maintaining the same swimming direction35. model are that it does not require the ability of sperm to Concomitantly, their swimming speed increases (this sense a descending chemoattractant gradient, and that phenomenon is known as chemo kinesis)17 as a result the sperma tozoa sample the environment intermittently of increased flagellar beat frequency35. Conversely, rather than continuously. when human spermatozoa are swimming away from Mammalian spermatozoa usually swim in either a the chemoattractant source bourgeonal, they abruptly straight or a curved line, depending on the pattern of turn around as a result of an asymmetrical flagellar flagellar movement42. However, little is known about beat and then swim towards the source35. This response to a descending chemoattractant gradient apparently differs from the presumed inabil- ity of sea-urchin spermatozoa to sense a descending Box 2 | Sperm transport in the female genital tract gradient of their chemoattractant, resact, owing to the In mammals that ejaculate semen into the vagina, sperm transport through the cervix irreversible resact–sperm binding41,43. This inability depends on sperm as well as on the muscular activity of the vagina, cervix and might also occur in human spermatozoa with the uterus90. In the uterus, the spermatozoa are passively driven towards the small opening chemoattractant (dissociation constant 22,90,91 of the oviduct (Fallopian tube) by waves of uterine smooth muscle contractions . × –10 REF. 44 Kd ≈ 6 10 M; ). Therefore, it is reasonable to This small opening at the uterotubal junction provides an additional barrier to sperm postulate that there are multiple mechanisms by which entry into the oviduct, and successful entry seems to require active sperm motility91. spermatozoa respond to a chemoattractant gradient, The number of spermatozoa that enter the oviduct is remarkably constant90 (in humans, only ~10% of the spermatozoa that were present in the uterus3). The oviduct has two depending on the species and the chemoattractant. regions, the isthmus and the ampulla (FIG. 1), each of which has different anatomical Another important question is how spermatozoa sense and physiological characteristics. The spermatozoa that enter the isthmus bind strongly the chemoattractant gradient. Do they compare the chemo- to the oviductal epithelium and become trapped, forming a reservoir91 (FIG. 1). It is attractant concentrations at different locations on the cell thought that, while residing in this storage site, the spermatozoa undergo membrane (the detection of a spatial gradient), or do they capacitation4,91, although asynchronously5,34. Owing to their lower affinity for the compare the chemoattractant concentrations at different epithelium, spermatozoa that become capacitated are released from the storage site. time points (the detection of a temporal gradient), as The few spermatozoa that are released probably require guidance to reach the ovulated do bacteria45? This question has not been addressed in 3 egg successfully , which, depending on the species, either resides in the isthmic– mammals. However, the finding that a sudden temporal ampullary junction or in the ampulla of the oviduct1 (FIG. 1). increase in the concentration of the chemoattractant

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Table 1 | Confirmed and putative chemoattractants for mammalian spermatozoa Substance Female source Species Reference Confirmed substances Atrial natriuretic peptide FF Human 59,60 Bourgeonal Unknown Human 36 Lyral Unknown Mouse 51 Peptides (<1.3 and ~13 kDa) FF Human 92 Progesterone* CO, FF, OF Human, rabbit 50 RANTES FF Human 54 Putative chemoattractants** Acetylcholine FF Mouse 93 Adrenaline FF Mouse 94 Antithrombin III FF Pig 95 Calcitonin FF Mouse 93 β-Endorphin FF Mouse 96 Heparin FF Human, mouse 97,98 Hyaluronic acid CO Human 99 Oxytocin FF Mouse 94 Peptide (8.6 kDa) FF Pig 21 Substance P FF Mouse 96 *Of the currently identified chemoattractants, only progesterone has been shown to be present near the egg in an aspirated human egg–cumulus complex50. **Substances that have been reported to cause sperm accumulation but have not been confirmed as chemoattractants. CO, cumulus oophorus; FF, follicular fluid; OF, oviductal fluid.

resact (achieved by the photorelease from caged resact — a fluid also occurs in vivo26, because the chemoattractant chemotactically inactive analogue of resact that releases gradient in the oviduct is anticipated to be maintained resact in response to a short pulse of light) causes a behav- for as long as the egg survives in the female genital tract ioural response of sea-urchin spermatozoa in the absence (~24 h post-ovulation in humans1). This would require a of a spatial gradient41,43 indicates that these spermatozoa continuous supply of chemoattractant, whereas the fol- sense a temporal gradient. licular fluid is only released as a single event at ovulation. Therefore, the physiological significance of the results Chemoattractants and their cellular origin obtained with follicular fluid is restricted to the implica- Over the years, many substances have been claimed to be tion that the egg and/or its surrounding cells secrete(s) a chemoattractants for mammalian spermatozoa11 (TABLE 1). chemoattractant(s) within the follicle prior to ovulation. However, because sperm accumulation at the optimal So, does the mammalian female egg–cumulus complex chemoattractant concentration — which is one of the secrete sperm chemoattractants subsequent to ovulation? characteristics of sperm chemotaxis — can be caused by This question has recently been answered by the findings other processes such as chemokinesis and sperm trap- that media conditioned either with individual, mature ping7,11, a clear-cut criterion for distinguishing between human eggs or with the surrounding cumulus cells are these processes (BOX 1) should be used in assays that iden- chemotactically active26. This indicates that sperm chemo- tify putative chemoattractants. So far, only a few putative attractants are secreted, not only prior to ovulation within chemoattractants satisfy the criterion discussed in BOX 1, the follicle, but also after egg maturation outside the follicle, and the discussion below is therefore restricted to these and that there are two chemoattractant sources: the chemoattractants. It should be noted that the physiological mature egg and the surrounding cumulus cells. significance of most of these substances is still unclear. Identification of mammalian chemoattractants. The Sources of chemotactic activity in mammals. One of the identity of the chemoattractant that is secreted from first physiological sources to be investigated for chemo- the egg is not yet known. One chemoattractant that is tactic activity was the mammalian follicular fluid7,11, secreted by the cumulus cells is probably progesterone. which contains the pre-ovulatory secretions of the egg and The identification of this steroid as a sperm chemo- its surrounding cells. Chemotaxis towards the follicular attractant was not straightforward. Initially, it was shown Photorelease fluid, which has so far been shown for human17, mouse14 that nM to mM concentrations of progesterone caused The rapid release of a 13 compound from its caged and rabbit spermatozoa, seems to be highly correlated the accumulation of human sperm, and that this could be 16 46–48 (protected) analogue by a short with the chance of fertilizing an egg, at least in humans . inhibited by a progesterone receptor antagonist . This pulse of light. However, it is unlikely that chemotaxis to the follicular and other findings led to the suggestion that progesterone

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Sea-urchin spermatozoa Human spermatozoa These findings have bearings on the issue of species CO ZP Resact Floral scent Progesterone specificity. A single chemoattractant that is common to Jelly Attractant various species would account for the lack of specificity Egg source? Egg in the chemotactic response of mammalian spermatozoa. Therefore, the secretion of progesterone by the cumulus cells could explain the lack of species specificity reported Resact [nM range] Bourgeonal [nM range] Progesterone [pM range] for follicular fluids and conditioned media of several mam- mals33. It remains to be seen whether the chemo attractant Receptor activation (guanylyl cyclase) OR17-4 activation ? that is secreted from the egg (which is probably a different Fast cGMP increase G-protein activation chemoattractant26) is species specific or common. Change in swimming direction (G ) at the midpiece olf Recently, two odorants — bourgeonal (at concen- 2+ –8 36 Transient [Ca in] increase trations ≥10 M) and lyral (at concentrations in the mAC activation 51 at the midpiece mM range) — were found to be chemoattractants for Asymmetrical beating human and mouse spermatozoa, respectively. As these [cAMP] increase, chemoattractants are probably not secreted in the female Change in swimming direction 2+ [Ca in] increase genital tract, it is likely that they are not the physiological sperm chemoattractants and that their cognate recep- Asymmetrical beating tors (discussed below) identify other, structurally similar chemoattractants (not yet known). Change in swimming direction Figure 3 | Models for the molecular mechanisms of sperm chemotaxis in marine The 8-kDa RANTES (regulated on activ- species and mammals. In the absence of sufficient molecular information, the models ation, normally T-cell expressed and secreted chemokine), of the molecular mechanisms of sperm chemotaxis are inevitably oversimplified. In the a potent chemoattractant for eosinophils, monocytes and case of spermatozoa of the sea urchin Arbacia punctulata, resact — a chemoattractant T lymphocytes52,53, was shown to be a chemoattractant that is produced by the egg jelly — binds to its specific surface receptor (probably for human spermatozoa. This chemo kine is known to be located on the flagellum), a guanylyl cyclase, and activates it. The resulting elevation of present in follicular fluid, and the mRNA for its recep- the level of cyclic GMP (cGMP) leads to a transient increase in the intracellular tor has been found in human spermatozoa54. RANTES is concentration of Ca2+ (Ca2+ ). This, in turn, causes the asymmetrical beating of the in produced by granulosa cells within the follicle in the ova- flagellum and, consequently, a change in the swimming direction. In the case of human ries, prior to ovulation, and its production is upregulated spermatozoa, the chemoattractant bourgeonal (or its physiological analogue) in some diseases that are associated with infertility54. activates the odorant receptor OR17-4, which is bound to a G protein (Golf) that is located at the midpiece of the flagellum. As a consequence, a membrane adenylate It is not known whether RANTES is also secreted in cyclase (mAC) is activated at the midpiece and the levels of cyclic AMP (cAMP) and the female genital tract after ovulation. If RANTES is 2+ a physiologically relevant chemoattractant for human Ca in rise. As before, these events induce the asymmetrical beating of the flagellum and, consequently, a change in the swimming direction. In the case of the sperma tozoa, it is an example of a substance that serves chemoattractant progesterone, which is produced by the cumulus cells, the signal- as a chemoattractant for different cell types. transduction pathway that is triggered by its binding to the progesterone receptor is Atrial natriuretic peptide (ANP) is a polypeptide not known. The location of the progesterone chemotaxis receptor in the hormone that is secreted from the atrial portion of the spermatozoon is also unknown. CO, cumulus oophorus; ZP, . heart and from other mammalian cell types, and that activates particulate guanylyl cyclase55,56. ANP is present in human follicular fluid57 and ANP-specific receptors have been identified on human spermatozoa58. Although was the chemoattractant in follicular fluid46. However, as sperm chemotaxis to ANP was demonstrated (but only discussed in BOX 1, the occurrence of sperm chemotaxis in the presence of a neutral endopeptidase inhibitor, such cannot be concluded on the basis of sperm accumula- as phosphoramidon, to avoid the inactivation of ANP by tion alone11, and therefore these results were inconclusive. the residual neutral endopeptidase-containing seminal Indeed, it was shown that progesterone is almost inactive fluid)59,60, it is not known whether ANP is involved in as a chemoattractant when used at the same concentra- sperm chemotaxis in vivo. tion as that found in chemotactically active follicular fluid, In the amphibian Xenopus laevis18, allurin is a 21-kDa and the accumulation of human spermatozoa caused by sperm chemoattractant. We mention it in the context of this steroid hormone was shown to be mainly owing to mammalian sperm chemoattractants because it shares sperm trapping, resulting from progesterone-stimulated homology with mammalian sperm-binding proteins61. hyperactivation49. However, progesterone was recently This chemoattractant is expressed and secreted from shown to be chemotactically active at concentrations as the X. laevis oviduct in a hormone-regulated manner. It A motility pattern that is low as 10–11–10–10 M for both human and rabbit sperma- is thought that at ovulation, as the egg progresses down characterized by increased tozoa, and evidence for a progesterone gradient along the the oviduct, allurin is applied onto the jelly layer that velocity, decreased linearity, 50 62 increased amplitude of lateral cumulus cell mass has been provided . (The reason why surrounds the X. laevis egg . Again, whether or not the head displacement, and spermatozoa chemotactically respond to progesterone at mammalian oviduct contains allurin-like chemoattractants flagellar whiplash movement. a concentration of 10–11–10–10 M, but not at much higher is an open question. concentrations16,49, is that, in chemotaxis, the response Granulosa cells The cells that form layers drops at too high chemoattractant concentrations; see Why are there so many chemoattractants? There are surrounding the oocyte within BOX 1.) It is not yet known whether the cumulus cells also several possible answers to this question. First, sperma- the follicle. secrete chemoattractants other than progesterone. tozoa might sense different chemoattractants; that

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is, they might undergo multistep chemotaxis as they in mammalian sperm74–82, and their localization to the travel through the female genital tract. Each step might midpiece of the tail of mature spermatozoa35,78, raised the sequentially guide the spermatozoa to the next chemo- possibility that some of these proteins might be chemo- attractant source. This implies that sperm guidance taxis receptors74,77. Recently, two distinct olfactory recep- might be much more elaborate than originally thought, tors — OR17-4 (also known as OR1D2) on the flagella and that chemotaxis extends over a much longer dis- of human spermatozoa36 and OR23 in mouse round tance. Second, perhaps each spermatozoon combines spermatids (exact location not yet determined)51 — were the information received from the different chemotaxis identified and their respective ligands, bourgeonal and receptors and ‘calculates’ its location according to the lyral, were found to be sperm chemoattractants. relative concentrations of the sensed chemoattractants. The stimulation of human spermatozoa with bour- 2+ 36 Third, it is possible that different chemoattractants geonal results in a transient rise of Ca in , originating elicit different behavioural sperm responses. Fourth, at the flagellar midpiece and propagating to the sperm because fertilization is so crucial, the multiplicity of head35. The finding of membrane-associated adenylyl chemoattractants might serve as a ‘back-up’. Finally, cyclase isoforms on the sperm flagellum and the different spermatozoa might respond to different observation that a specific antagonist of this enzyme chemoattractants, resulting in sperm selection. This (SQ22536 at a concentration of 5 mM) inhibited the possibility is consistent with the suggestion (based chemotactic response to bourgeonal, raised the pos- on the finding that human leukocyte antigen-linked sibility that the chemotactic response to bourgeonal olfactory receptor genes are transcribed in the testis) that might be mediated by membrane adenylyl cyclase35. (To testis-expressed human leukocyte antigen and olfactory substantiate this possibility, experiments demonstrating receptor proteins are functionally connected and serve that bourgeonal indeed causes cAMP elevation, and that 2+ in the selection of spermatozoa, enabling them to dis- such elevation increases Ca in, should be carried out tinguish ‘self’ from ‘non-self’63. It is also possible that with human spermatozoa.) It therefore seems possible chemoattractant-specific sperm selection, if it exists in that the chemotactic stimulation of OR17-4 triggers a mammals, might be involved in sperm competition or signalling pathway similar to that of the olfactory sys- might enable the female to choose sperm. We would like tem35 (FIG. 3). It should be pointed out that many of the to emphasize that all these possibilities are speculative olfactory receptors are expressed in the testes; therefore, and there is currently no hard evidence in support of it is quite possible that many of them are simultaneously any of them. present in sperm. If this is found to be the case, it will be in line with the multiplicity of chemoattractants, as Molecular mechanisms of sperm chemotaxis discussed above. The molecular mechanisms of sperm chemotaxis The finding that progesterone at concentrations in are poorly understood. The few known pieces of the pM range is a chemoattractant for human and rab- the puzzle were derived from studies in marine bit spermatozoa 50 indicates that at least one of the two invertebrate spermatozoa, in which sperm chemo- progesterone receptors, which were identified on the cell attractants are well known and in which (unlike surface of mammalian spermatozoa44,83,84 (and that are mammals) most of the spermatozoa are chemotact- distinct from the nuclear progesterone receptor of somatic ically responsive and, therefore, sperm chemotaxis cells), is a chemotaxis receptor. These progesterone recep- 9,10 × –10 × –5 is easily measurable . Models of the molecular tors bind progesterone with a Kd of 6 10 and 3 10 M, mechanism of sperm chemotaxis in such species have been respectively44. They have not yet been cloned, isolated or published (for reviews, see REFS 64,65). However, recent sequenced. One of the progesterone receptors is located studies, carried out primarily in the sea urchin Arbacia at the head of the spermatozoon, but it is not known punctulata43,66 and the starfish Asterias amurensis67, whether this is the chemotaxis receptor or whether the do not substantiate the published models. These studies progesterone chemotaxis receptor is a different protein led to the proposal of another model, in which the located on the flagellum, similar to OR17-4. chemotaxis receptor, a guanylyl cyclase68 located on The signal-transduction pathway that takes place dur- the flagellum67, is activated in response to chemo attractant ing chemotaxis to progesterone has not been elucidated. binding69 (resact in A. punctulata70 and asterosap in Recently, however, Harper et al.85 showed that a concentra- A. amurensis67). Consequently, the concentration of cyclic tion gradient of progesterone, starting at less than 10–8 M, Olfactory receptor 43,67 2+ An integral membrane protein GMP rises rapidly and transiently , and this transient stimulates Ca in oscillations at the junction between the that is associated with a rise is thought to result in a spike of the intracellular caudal head and the midpiece of the spermatozoon. These G-protein and is involved in 2+ 2+ 43,67,71,72 2+ 2+ concentration of Ca (Ca in) . The Ca in elevation Ca oscillations, which are reminiscent of the faster oscil- effecting the sense of smell. is known to bring about increased flagellar asymmetry latory spikes observed in the sea urchin A. punctulata and reduced linearity of swimming71,73. So, depending on stimulation with the chemoattractant resact41, are Spermatid 2+ An immature gamete that on the timing of the Ca in spikes, sperm swimming is synchronous with flagellar activity, indicating that 41 85 develops into a spermatozoon. altered to approach the chemoattractant . they modulate the flagellar beat . Other progesterone- The available information about the molecular triggered molecular events have been identified in Nuclear progesterone mechanisms of sperm chemotaxis in mammals is limited mammalian spermatozoa (for example, cell membrane receptor A progesterone-inducible and mainly includes the identity of some chemotaxis depolarization, increased intracellular pH, elevated 2+ transcription factor that is receptors and chemoattractant-induced Ca in changes. cAMP concentration, and the activation of phospholi- located intracellularly. The finding of G-protein-coupled olfactory receptors pase A, protein kinase C and other kinases). However,

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because progesterone stimulates a number of other much larger than the distance employed in the in vitro sperm functions, such as hyperactivation, capacitation thermotaxis assay8, the temperature gradient that is and acrosome reaction, most of these molecular events sensed by the spermatozoa in vivo is probably shallower. were associated with these functions (see REF. 86 for a It is not yet known whether spermatozoa can sense as review). It remains to be seen whether some of these shallow a temperature gradient as the one that exists within intracellular events are also part of the progesterone- the oviduct. As in the case of mammalian chemotaxis, only induced chemotactic signalling pathway. capacitated spermatozoa are thermotactically respon- Another possibility is that, as in sea-urchin and starfish sive8. The molecular mechanism of sperm thermotaxis spermatozoa, one of the chemotaxis receptors on mam- is not yet known. malian spermatozoa is guanylyl cyclase. However, the only indirect evidence for this is the finding that human Conclusions and perspectives spermatozoa respond chemotactically to ANP, which is The research field of mammalian sperm guidance was an activator of membrane guanylyl cyclase59. It is possible non-existent a little more than a decade ago. In recent that mammalian spermatozoa possess several signal- years, the concept of mammalian sperm guidance has transduction pathways that might be complementary, or been revolutionized in the sense that, unlike the prevailing that different chemoattractants might trigger different dogma, it became clear that mammalian spermatozoa signal-transduction systems. have the ability to be actively guided to the egg. Two of the mechanisms by which this guidance is achieved have Mammalian sperm thermotaxis been revealed: chemotaxis and thermotaxis. However, The fertilization site in the oviduct has been shown to many important questions are still unresolved. For be 1–2°C warmer than the storage site for spermato- example, what are the identities of all the physiological zoa in rabbits28 and in pigs29 (FIG. 1). However, data for chemoattractants? Are the egg and the cumulus cells the the temp er ature in the human fallopian tube are not only physiologically relevant sources of chemoattractants available. A recent study in rabbits showed that this in vivo? Are there sequential processes of chemotaxis temperature difference is time dependent and that the along the oviduct? Are there several different chemo- difference increases from 0.8 ± 0.2°C before ovulation attractant-specific, behavioural response mechanisms in to 1.6 ± 0.1°C after ovulation24. This change is due each mammalian species? And if so, what purpose does to a temperature decrease at the storage site rather than this multiplicity of mechanisms serve? What are the a temperature increase at the fertilization site. Three signal-transduction pathways that are involved in sperm mechanisms have been proposed for the ovulation- chemotaxis in response to the various chemoattractants, dependent temperature decrease at the storage site24: and do these pathways function in concert or individually first, the hormone-controlled localized release of acid under different conditions? Does thermotaxis have a mucus glycoprotein — a macromolecule that undergoes unique signal-transduction pathway or does it converge extensive endothermic hydration — at the storage site87; on one of the chemotaxis pathways? What is the identity second, blood from the nearby ovarian vein cools the of the thermosensor for thermotaxis? Furthermore, all blood that enters the storage site by counter-current the conclusions about the function and location of sperm heat exchange28,29,88; and third, an ovulation-dependent chemotaxis and thermotaxis have been reached on the change in the source of blood supply to the storage site basis of in vitro experiments only and they, therefore, — the warmer ovarian artery prior to ovulation and await verification in vivo. the cooler uterine artery subsequent to ovulation89. It is Obtaining answers to questions like these will not only possible that these mechanisms function in concert to increase our understanding of mammalian fertilization reduce the temperature at the storage site efficiently. but might also allow obvious clinical applications. For Bahat et al.8 showed that rabbit and human sperma- example, both chemotaxis and thermotaxis can potent- tozoa can sense a temperature difference and respond to it ially be used in clinical procedures to obtain sperm by thermotaxis; that is, by swimming from the cooler to populations that are enriched with capacitated sperma- the warmer temperature. The thermotactic response is as tozoa in vitro. They can also be exploited as a diagnostic strong at a temperature difference of 0.5°C as at a differ- tool to assess sperm quality. In addition, these processes ence of 2°C, which indicates that spermatozoa can sense can potentially be used, in the long run, as a means small temperature differences. However, because the of contraception by interfering with the normal process of distance between the storage and fertilization sites is fertilization.

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