Sperm in the Mammalian Female Reproductive Tract: Surfing Through the Tract to Try to Beat the Odds

Abstract

Mammalian sperm are deposited in the vagina or the cervix/uterus at coitus or at artificial insemination, and the fertilizing sperm move through the female reproductive tract to the ampulla of the oviduct, the site of fertilization. But the destination of most sperm is not the oviduct. Most sperm are carried by retrograde fluid flow to the vagina, are phagocytosed, and/or do not pass barriers on the pathway to the oviduct. The sperm that reach the site of fertilization are the exceptions and winners of one of the most stringent selection processes in nature. This review discusses the challenges sperm encounter and how the few sperm that reach the site of fertilization overcome them. The sperm that reach the goal must navigate viscoelastic fluid, swim vigorously and cooperatively along the walls of the female tract, avoid the innate immune system, and respond to potential cues that provide directionality to their movement.

1. INTRODUCTION

After semen deposition, sperm immediately encounter challenges to reach the site of fertilization. These challenges include the distance to reach the oocyte, the many folds of the female tract, retrograde fluid flow, acidic conditions in the vagina, mucus in the cervix and viscoelastic fluid through the tract, immune responses to sperm, and other barriers that are incompletely understood. Due to these challenges, most of the millions or billions of sperm that are deposited in the female reproductive tract never reach the site of fertilization. Only about 0.001 to 0.1 % of the sperm that are deposited are found in the upper oviduct near the time of fertilization (1–3). Are the sperm that can reach oocytes selected in some way during the journey? What traits do the selected sperm possess? Are the “fittest” sperm the ones that arrive in the upper oviduct for fertilization? Are those that reach the site of fertilization simply in the right place at the right time? Or is there some combination of fitness selection and good fortune that decides which sperm fertilize oocytes? This review will attempt to address these questions and will focus on studies carried out in mammals.

The movement of the sperm that are successful in fertilizing oocytes has been complicated to study because the majority of the sperm are not the cells of interest; that is, most sperm do not reach the site of fertilization and their movement may obscure observations of sperm that reach the oviduct. Aside from some overt features such as normal motility and proper morphology (3), at a cellular level, one cannot distinguish the fertilizing sperm or the sperm that reach the site of fertilization from the sperm that do not.

At mating, in many species, males deposit semen in the vagina. A fraction of the vaginal sperm moves into and through the cervix into the uterus (Figure 1). In contrast, males of some species deposit semen into the cervix and, due to the small volume of the cervix compared to the relatively large volume of semen, the semen moves immediately into the uterus. During routine artificial insemination in most species, semen is deposited in the uterus, bypassing the vagina and cervix. Once in the uterus, a small proportion of the uterine sperm moves through the utero-tubal junctions into either of the oviducts. The opposite end of the oviduct also collects ovulated oocytes and transports them to the ampulla.

Figure 1.

Model of how sperm move through the female reproductive tract. Sperm are deposited into either the vagina or cervix–uterus. If deposited in the vagina, sperm must move quickly from the vagina into the cervix and uterus. In the cervix, as pusher microswimmers, they are directed to swim along the deep grooves where they can move more rapidly and avoid retrograde mucus flow. In the uterus, contractions move sperm to the uterotubal junction, although many sperm are phagocytosed or lost in retrograde flow. At the uterotubal junction, sperm may cluster so that they can swim synchronously to pass into the isthmus, where some sperm adhere to glycans on the epithelium and others are carried by peristaltic fluid waves. When released from the isthmic epithelium, sperm can advance in the grooves of the isthmus to the ampulla, where the narrower grooves affect sperm motility. In the ampulla, they can fertilize mature oocytes.

It is important to note that, when semen is deposited by natural mating, in addition to sperm, seminal fluid is deposited, which contains bioactive components that can act on the vagina, cervix, and uterus (4). But with the progression of sperm closer to the site of fertilization, seminal fluid is further diluted and fewer seminal fluid components are brought with advancing sperm. It is likely that very few seminal fluid components are brought to the oviduct (5). During artificial insemination, the medium or extender into which semen is diluted is also deposited and the seminal fluid is removed or diluted. But like seminal fluid, little of the extender is expected to be carried into the oviduct.

2. APPROACHES FOR STUDYING SPERM TRANSPORT IN THE FEMALE REPRODUCTIVE TRACT

Sperm transport has been studied using several approaches that are discussed below. Some techniques can only be applied to oviducts because the walls of the other parts of the female reproductive tract are too thick to observe sperm within.

2.1. Collection of Regions of the Female Tract after Insemination

The first studies of sperm transport were done by collecting different anatomical regions of the female reproductive tract at various times after mating or artificial insemination. These studies provided a snapshot of the location of sperm. In most studies, sperm were collected for enumeration by flushing the region of the tract and also examining the flushed tract for retained sperm (3). Although these studies provided useful information, with this approach, the dynamic nature of sperm movement in the reproductive tract could not be investigated. The other caveat is that it is difficult to be certain that the sperm bound in deep folds and grooves are removed by flushing or are not missed during the enumeration of sperm remaining bound to the tract.

2.2. Digital Live Cell Imaging in Oviducts

Bovine, porcine, and mouse oviducts have been studied in situ under a microscope after opening them longitudinally and fixing them in place (6, 7). This approach measures ciliary activity and the movement of sperm, oocytes, and embryos on the surface of the exposed epithelium. High-quality images can be obtained but the major limitation is that, because the oviduct is opened and medium is added, fluid movement due to smooth muscle contractions is disturbed.

2.3. Observing Labeled Sperm by Fluorescence Microscopy

Events in the female reproductive tract have been observed in rodents by exteriorizing the tract and placing it in medium at body temperature. Recent experiments using mice have used sperm genetically tagged with two fluorescent proteins to allow the use of fluorescence microscopy of live cells. Two genes were knocked-in into mice, the eGFP gene controlled by the acrosin promoter produced green fluorescent protein in the acrosome, and the DsRed2 gene attached to a gene encoding a mitochondrial protein (8). In this scenario, under a fluorescence microscope, all sperm fluoresced red in the midpiece, and those with intact acrosomes were also labeled green (9, 10). Sperm could be observed in both the uterus and oviduct.

For larger animals, a unique approach using fibered confocal microscopy has been applied to exteriorized tracts (11). Fluorochrome-labeled sperm were deposited in the ewe uterus and confocal fiberoptic probes of varying sizes were inserted individually into the lumen of the tract. Fluorochrome-labeled sperm could be observed at a resolution that could identify sperm and assess their motility.

2.4. Observing Unlabeled Sperm in the Oviduct by Optical Coherence Tomography (OCT)

The fluorescence microscopy experiments are limited to 2-dimensional observations making it difficult to track sperm that move in and out of the Z-axis rapidly. Recently, OCT has been applied to observe sperm and oocytes 3-dimensionally at micrometer resolution in vivo in the oviduct (12). OCT can be applied to live tissues and does not require sample preparation or ionizing radiation. Because OCT collects images to only a depth of 1–2 mm below the surface, a “window” was attached to mice for OCT observations of the oviduct (12, 13). Using this technique, sperm appear as bright spots in the image, as do cells and other particles. But the frequent change in the direction of motile sperm can be used to distinguish sperm from other bright spots in the oviduct.

3. SPERM MOVEMENT THROUGH THE FEMALE REPRODUCTIVE TRACT

3.1. Speedy Movement out of the Vagina

The semen of primates including humans, ruminants, rabbits, and many other animals is deposited in the anterior portion of the vagina, near the cervical opening (14, 15). There is a rapid infiltration of neutrophils into the vagina in response to semen deposition (16, 17). The pH of the thin-walled vagina is acidic, which is toxic to pathogens and prevents sperm from surviving for more than short periods. The acidic pH is primarily due to lactic acid produced by the dominant genus of microbes, Lactobacillus (18). Lactobacilli help maintain a healthy female reproductive tract by producing bactericidal substances and occupying an ecological niche. In contrast, dysbiosis of the vagina allowing, for example, Escherichia coli to colonize the vagina can reduce fertility through membrane lipid peroxidation (19) and by immobilizing sperm (20, 21). But, despite the beneficial effects of Lactobacillus, for sperm to survive, they must quickly exit the vagina and move into the cervix where the pH is more neutral. In humans and rodents, sperm move out of the pool in the anterior vagina and into the cervix within minutes (15, 22). In rodents, secretions from accessory sex glands in semen remain in the vagina forming a plug that caps the cervix, blocking sperm from additional males from passing into the cervix and semen retrograde flow out of the cervix (23, 24). Interestingly, in rodent species in which females mate with more than one male in a brief period, males have larger accessory glands that presumably produce larger, more effective cervical plugs, possibly enabling the first male to have a selective advantage over rivals (25).

3.2. Sperm Movement Through the Grooves of the Cervix

The cervix hinders the movement of microorganisms from the vagina but allows sperm passage (26). In contrast to the vagina, the cervical architecture is much more complex (27). The thick walls of the cervix contain longitudinal folds that produce deep grooves where many sperm are found shortly after mating (28). The cervix produces a highly viscous fluid containing abundant mucin glycoproteins. Immunohistochemical staining revealed that deep grooves of the cervix from animals in the follicular stage had more negatively-charged sialylated mucins and the regions near the center of the lumen had more neutral and sulfated mucins (28). The observation that the deep grooves contained sperm and that these sperm were oriented toward the uterus suggested that advancing sperm moved through the deeper grooves. Fluid flows toward the vagina and cervical fluid in the grooves moves slower than in the lumen, leading to the hypothesis that the faster flow in the center of the lumen may flush pathogens back into the vagina and outward (29).

A second reason that sperm in the cervix are also found in the grooves is the tendency of sperm to swim adjacent to a solid-liquid interface because their tail beat pattern is that of a “pusher microswimmer” (30, 31). When pusher microswimmers reach a wall, the 3-dimensional rotating beat pattern of their tail points them in a direction along the wall (32) resulting in movement that hugs the wall. Sperm accumulate to an even greater extent in the 90° corners of microchannels (33–35). Human sperm, when within 1 μm of a wall, also intermittently change the tail beat pattern from a 3-dimensional pattern to a 2-dimensional “slither swimmer” pattern in higher viscosity medium (36). Sperm in the slither swimmer pattern do not rotate, in contrast to the normal 3-dimensional tail beating. All these characteristics are expected to increase the tendency of sperm to swim along the walls of deep grooves in the cervix rather than in the middle of the lumen so that sperm can advance into the uterus (Figure 2A). Human sperm swimming along a solid surface also swim faster and straighter in slither mode. Sperm found in groups swimming in slither mode had more tightly synchronized tail beat patterns (36). Thus, sperm behavior along the walls of the cervical grooves is likely very different than in the lumen and is important for progression in the female tract.

Figure 2.

Sperm movement through folds of different sizes in the female reproductive tract. Sperm move along the walls of the tract and accumulate in the corners and deep grooves, a characteristic of pusher microswimmers. Their motility pattern keeps them near the walls, and if they stay within 1 μm of the wall, they will sometimes develop a slither-swimmer motility pattern, enabling them to progress faster. The folds have larger spaces between them (a) in the isthmus than (b) in the ampulla. The restricted space in the ampulla affects the motility pattern of sperm between the narrower folds.

In addition to the influence of walls on sperm behavior, the fluid viscosity can change the tail beat pattern. Changes in cervical fluid viscosity and volume during the estrous cycle have been well documented (37). A viscoelastic fluid (29, 38), cervical mucus is difficult for sperm that have sluggish or abnormal motility to swim through. Thus, the cervix selects more vigorously motile sperm. Microfluidic devices containing viscoelastic medium have been used to select sperm with improved viability and motility (39).

After semen deposition, neutrophils begin to infiltrate the lumen of the cervix (40). This infiltration is more limited than what occurs in the uterus (40). Surprisingly, this infiltration may not be a major hindrance to sperm because when female rabbits were mated to a second male after the cervix had mounted a leukocytic response to the first mating, sperm from the second male were still able to fertilize oocytes (41). This suggests that sperm that reach the site of fertilization may not remain in the cervix long enough to be phagocytosed by neutrophils.

3.3. Sperm Movement Through the Uterus: Surfing, Phagocytosis, and Possible Sperm Storage

3.3.1. Uterine smooth muscle contractions.

Sperm movement through the uterus is not by their own motility but primarily by “surfing”, that is, being carried in the fluid that is propelled by uterine contractions. The uterine lining includes the myometrium, a layer of smooth muscle capable of producing strong, coordinated contractions. Uterine contractions in the direction of the ovary are strong during estrus (42). These contractions move uterine fluid which carries sperm toward the oviduct. In a classic experiment, 5–40 μm technetium-labeled macrospheres were placed at the external cervical opening of women and detected with sonography so that fluid flow could be observed (43). If the macrospheres were deposited in the early follicular phase, only a few were found in the oviduct but if deposition occurred in the late follicular phase, many more were observed in the oviduct. This demonstrates that the directional coordination of uterine contractions changes near ovulation. Transport from the cervical opening to the oviduct was rapid; macrospheres were recovered in the oviduct as early as 1 min after deposition. Measurements of uterine contractility in cows and ewes also indicate that contractile activity increases near estrus and is oriented towards the oviduct (42).

Experiments using fibered confocal microscopy on exteriorized ewe tracts described above confirmed that after semen deposition, sperm accumulated at the tip of the uterus near the UTJ (11). Ram sperm were observed in the base, middle, and tip of the uterus, the UTJ, and the oviduct isthmus. The high-resolution video images of sperm showed that the tip of the uterine horn had the greatest sperm concentration 4 hr after insemination and that the UTJ was a functional storage site (11). Thus, most sperm in the uterus at that time were near the UTJ.

In species such as pigs in which semen is deposited directly into the uterus, the liquid components of semen may enhance uterine contraction (44, 45). Thus, dilution of semen in an extender used for artificial insemination might dilute active components in seminal fluid. Interestingly, there is some evidence in swine that supplementing semen extenders with seminal fluid (46) or the hormones found normally in semen such as estrogens and other hormones such as prostaglandin F_2α and oxytocin reduces fertility depression during the summer months (47–49).

Even though uterine contractions are coordinated to move fluid and sperm toward the oviduct, there is still a considerable loss of sperm due to outward flow through the cervix and vagina. For example, in cattle, even when sperm are inseminated directly into the uterine body, most sperm are lost by retrograde flow and phagocytosis rather than advancing into the oviduct (3, 42).

3.3.2. Immune response to semen in the uterus.

Sperm in the uterus generate a rapid and transient innate immune response activating infiltration of neutrophils and macrophages, an observation made in many species (50–52). Bovine, porcine, and human sperm induce PMN infiltration and the formation of neutrophil extracellular traps (NETosis) within a couple of hours after insemination that can eliminate microorganisms and trap sperm (53–55). At least in cattle, activation of the innate immune system occurs mainly through toll-like receptor 2 (TLR2) signaling (56). Although trapping sperm would seem to be undesirable, the sperm remaining in the uterine lumen, at least a few hours after insemination, may be abnormal and removal may be beneficial. Due to the tendency for sperm to swim near walls, a high proportion of sperm may be found near the uterine epithelium. Sperm also can bind directly to the uterine epithelium in species such as swine (5, 57). The sperm bound to the epithelium have normal ultrastructure and high mitochondrial membrane potential but sperm that are free in the lumen have more abnormalities (58). The binding of higher fertility sperm to the uterine epithelium suggests that the uterus may be a site of sperm storage (57). Retention near the epithelium may protect sperm from NETosis. However, studies in pigs in which the utero-tubal junction was ligated 1–2 hr after mating indicated that sperm in the oviduct within 1–2 hr gave the maximum fertilization (59). Thus, the fertilizing sperm appear to spend little time in the uterus.

In addition to the immune response generated by sperm, there are a variety of reports demonstrating that sperm-free seminal fluid induces leukocyte infiltration and induction of cytokine gene expression (60–63) but other reports suggest an immunosuppressive role of seminal fluid (64). Surprisingly, the NETosis response to the liquid portion of the inseminate is greater than the response to sperm (53). The response appears to be due to components in the fluid as well as simply a general response to any liquid. Simply depositing a volume of liquid into the uterus of pigs, regardless of the components of that liquid or whether it contains sperm or seminal fluid, can activate neutrophil infiltration (65) although not to the same degree as semen.

3.3.3. Sperm entry into the oviduct by the utero-tubal junction (UTJ).

For sperm to move from the uterus to the oviduct, they must pass through the utero-tubal junction (UTJ). In addition to the cervix, the UTJ appears to be a significant barrier to the advancement of sperm to the site of fertilization. For animals that deposit semen in the uterus at coitus or at artificial insemination, the UTJ is the single major barrier for sperm to reach the oviduct. The structure of the UTJ varies across species; it is lined with a species-variable number of folds that may serve to control sperm entry into the usually small lumen (9, 22). Its secretion appears viscoelastic, due to the abundant mucus. Viscoelasticity includes both viscosity (thickness) and elasticity, the ability of a material to reform its original shape, such as the shape of a container. Swimming through a viscoelastic fluid is a challenge for sperm (31).

In mice, there is evidence that the UTJ gives preferential passage to sperm with less fragmented DNA (66) but the mechanism by which this selection occurs is unclear. But the UTJ seems to be the location of the most stringent sperm selection. There are reports of mice that have targeted mutations in many genes such as ADAMs (proteins that contain Adhesion, Disintegrin, and Metalloprotease domains), molecular chaperones, etc. that produce sperm with normal motility and morphology that are unable to pass through the UTJ (67). Many but not all of these mutations affect ADAM3 expression and localization on the sperm surface (67, 68). However, ADAM3 is a pseudogene in humans (69); thus, ADAM3 function is not required in all mammals.

The recent observation that sperm formed clusters in the uterus in regions where there are crypts and in the UTJ, with their heads and tails oriented in the direction of the oviduct, suggested that clustering may be important for movement through the UTJ (70). When the number of sperm inseminated was reduced, the number of sperm in each cluster was also reduced. The requirement for high sperm numbers to form clusters may partly explain why so many more sperm must be deposited at mating or insemination than are necessary for normal fertility. Finally, the authors found that sperm from Tex101 null mice only formed very small groups with disordered orientation (70). Even when both wild-type and Tex101^−/− sperm were present in the female tract, only wild-type sperm formed clusters and passed through the UTJ. In viscoelastic medium, wild type and Tex101^−/− sperm displayed different swimming patterns that might explain why Tex101^−/− sperm cannot form clusters. They also observed that in clusters, sperm tails waved in a synchronized manner, which may generate a more forceful push to “open” the UTJ. It is known that fluid with high viscoelasticity like that found in the UTJ can induce bovine sperm to form clusters that align in the same direction and swim cooperatively in a more progressive fashion in the absence of fluid flow or upstream against an intermediate flow (71). Transient pulses of fluid flow are most effective at forming individual sperm into clusters (72).

3.3.4. Sperm in the oviduct.

Once sperm traverse the UTJ, they enter the oviduct, an elaborate tube with multiple important functions. Recent 3-dimensional imaging studies of the mouse oviduct have illustrated the anatomical complexity of the fold patterns (73). The mouse oviduct has consistent folding patterns that correspond to cellular differences in the epithelium of the oviduct. For example, the abundance of multiciliated cells does not decrease gradually moving through the ampulla and isthmus but instead decreases significantly between loops 2 and 3 of the ampulla. At the cellular level, additional complexity has been revealed by studies that used single-cell RNAseq, revealing as many as 12 cell types compartmentalized into stromal, epithelial, and immune cells (74–78). Analysis of the epithelial subtypes yielded 4 ciliated and 6 non-ciliated secretory cell subtypes (74). The transport of sperm, oocytes, and embryos appears to be mediated by a combination of smooth muscle contraction and movement of fluid, by ciliary activity and movement of fluid by the cilia, and by the production and flow of secreted fluid (79).

3.3.5. Sperm in oviduct fluid.

The first part of the oviduct (closest to the uterus) is the isthmus. A study of the exteriorized mouse oviduct and fluorescent protein-labeled sperm demonstrated that some sperm are bound to the isthmus and others are moved alternately in an ovarian and uterine direction. This movement back and forth resembles peristalsis and can be inhibited by the addition of the anticholinergic drug prifinium bromide, an inhibitor of peristaltic contraction (10). Even though there is considerable back-and-forth movement, studies labeling fluid flow with India ink showed a net fluid flow in the oviduct in the direction of the ovary, an important observation when considering sperm taxis, below (80).

Even with the net fluid flow in the ovarian direction, studies from several groups found that sperm were much more abundant in the isthmus than in the ampulla at all times examined (0.25 – 8 hr post-coitus in the mouse) (9, 81, 82). Interestingly, the few sperm in the ampulla were mostly acrosome-reacted, in agreement with the report that mouse sperm that fertilize oocytes begin the acrosome reaction before entering the oocyte cumulus matrix (83). The trigger for the acrosome reaction in the oviduct is uncertain.

3.3.6. Sperm bound to the oviduct epithelium forming a reservoir.

In addition to sperm suspended in oviduct fluid, many sperm bind to the epithelium of the isthmus, forming a storage reservoir (84). Some of the first demonstrations that sperm adhere to the oviduct epithelium were done by Ron Hunter, who resected the isthmus in rabbits and pigs and observed an increase in polyspermy, presumably because excess sperm advanced to the ampulla to fertilize oocytes (85, 86). Thus, by retaining and storing sperm, the isthmus acts as a “buffer” of sperm number and reduces polyspermy.

As part of its sperm storage function, the isthmus increases sperm lifespan. Binding to oviduct epithelial cells maintains sperm viability and fertilizing capacity compared to incubation in medium or with tracheal epithelial cells (87–89). Oviduct cells may accomplish this by suppressing capacitation and Ca²⁺ influx in sperm (90, 91).

Sperm retention in the isthmus and suppression of Ca²⁺ influx both required sperm contact with oviduct epithelial cells (87–91). Attempts to identify receptors first centered on oviduct epithelial cell carbohydrates (glycans) due to the noted ability of sperm to bind glycans. The initial work was done by testing monosaccharides or a few simple oligosaccharides for their ability to bind sperm and inhibit sperm binding to isthmic epithelial cells or explants. Several different monosaccharides including mannose, galactose, and fucose inhibited sperm binding to oviduct cells obtained from a variety of species in competition assays (92). The conclusions from many of these experiments must be taken with caution, however, because usually high concentrations were required that may be toxic, and proteins that bind glycans often recognize more than a simple monosaccharide. But the development of printed glycan arrays (Figure 3A) allowed the screening of hundreds of insoluble glycans for their ability to bind proteins (lectins) (93, 94). Using that information, glycan motifs that lectins bind could be identified. The same approach was applied to porcine sperm that were tagged with a fluorochrome so that bound sperm could be detected. Using this approach, the first detailed screen of about 400 glycans identified structures that bind porcine sperm with exquisite specificity. These structures all contained either of 2 specific smaller glycan motifs, a 6-sialylated biantennary N-linked glycan, and (Galβ1–4[Fucα1–3]GlcNAcβ) the Lewis X trisaccharide (95, 96). Porcine sperm binding to these glycans is remarkably specific. Even though porcine sperm bind 6-sialylated biantennary glycans, they do not bind 3-sialylated biantennary glycans; the linkage of sialic acid is critical. Moreover, porcine sperm do not bind Lewis A trisaccharide (Galβ1–3[Fucα1–4]GlcNAcβ), a positional isomer of Lewis X (Figure 3B).

Figure 3.

Identification of glycans that bind sperm using an array of glycans. (a) A variety of glycans were spotted on a microscope slide forming an array. Sperm were stained with Hoescht and added to the array. After washing the free sperm from the array, it was scanned to identify the glycan spots that bound sperm. The inset shows an enlargement of one spot that bound sperm. (b) Structures of glycans that bound porcine sperm on the array and related structures that did not. Le^X and suLe^X bound porcine sperm, but the isomers Le^A and suLe^A did not. The branched 6-sialylated oligosaccharides (bi-SiaLN) also bound porcine sperm, but the 3-sialylated structures did not (not shown). The branched galactoside (bi-LN) bound fewer sperm than the 6-sialylated structures but many more than N-acetyllactosamine (LN), a disaccharide found in Lewis trisaccharides and the branched structures. Panel A provided by Leonardo Molina. Panel B adapted with permission from reference 97 CC BY4.0.

Both glycan motifs are found in the porcine isthmus. 6-sialylated glycans are very abundant throughout the entire oviduct and Lewis X glycans are abundant in the isthmus although much less abundant in the ampulla (95, 96). Each glycan motif is sufficient to bind sperm and required for robust binding. Masking each oviduct glycan reduces sperm binding to oviduct cells in vitro to a maximum of 60%; they are partially but not completely redundant sperm receptors. If either glycan motif is immobilized onto beads, sperm bound to the beads have a longer lifespan, mimicking what binding to oviduct cells accomplishes (97). The longer lifespan may be due to glycan suppression of the normal increase in intracellular Ca²⁺ that occurs during capacitation (97). There is also evidence that the longer sperm lifespan is due to metabolic changes induced by binding oviduct glycans. Targeted metabolomics experiments demonstrated that Lewis X transiently diminishes the abundance of the immediate ubiquinone precursor and suppresses the formation of the citric acid cycle component fumarate (98). This diminishes the activity of the electron transport chain reducing the production of damaging reactive oxygen species (98). The mechanism by which oviduct glycans affect sperm metabolism is unclear but of great interest, particularly considering that sperm from animals such as some species of bats or insects are stored for months or years (99).

The observation that sperm from a variety of species have glycan binding preferences that differ between species is intriguing. While most studies that have drawn these conclusions have not identified glycan structural features beyond the terminal monosaccharide, the terminal monosaccharide identified as important for a particular species is quite variable (100). For example, galactose inhibited equine sperm binding to oviduct cells (101), fucose inhibited bovine sperm (92) and sialic acid inhibited binding to hamster sperm (102). The few studies that have identified more structural features have yielded interesting results. Lewis X (Le^X), particularly the sulfated form, was identified as one glycan motif that binds porcine sperm (95, 96). Bovine (Bos taurus) sperm do not bind Le^X but instead, bind Lewis A (Le^A). The sulfated version of Le^A also binds more bovine sperm that the unsulfated version (103).

The close relationship of two glycans that bind sperm from two ungulates suggests a possible evolutionary relationship among sperm-binding glycans. More distantly-related species may bind more unique oviduct glycans. This has implications for the reproductive isolation of species. Reproductive isolation is enforced by barriers that act at different stages: 1) premating, 2) post-mating-prezygotic, or 3) postzygotic (104). Premating isolation is caused by biological, including both behavioral and anatomical, traits that prevent populations from encountering or mating with each other. Post-mating-prezygotic barriers may be due to incompatibility between gametes or between sperm/seminal fluid and the female reproductive tract (i.e. sperm storage in the female tract)(105). Little is known of mechanisms that may be responsible for post-mating, pre-zygotic species isolation (PMPZ) but in the Drosophila virilis species group, PMPZ is due largely to defective sperm storage after copulation (106). Among vertebrates, PMPZ and defective sperm storage may play a role in speciation, but almost no studies have tested this possibility (107, 108). The specific oviduct glycans important for the formation of the sperm reservoir may be shared only among closely related species.

3.3.7. Suppression of the immune response in the oviduct.

In stark contrast to the innate immune response to sperm activated in the cervix and uterus, in the oviduct sperm do not trigger a phagocytic response and can survive for extended periods ranging from hours to months, depending on the species (99, 109, 110). Indeed, in the isthmic region of the oviduct (nearest to the uterus), the presence of macrophages or neutrophils is rare (111). The oviduct appears to confer unique immunological privilege within the female reproductive system that enables sperm to avoid elimination by phagocytes.

Recent data indicated that oviduct immune privilege may be stimulated by the presence of sperm. The oviduct is not broadly immune-inhibited because the innate and adaptive immune system can effectively clear pathogenic bacteria that cause disease and infertility in humans (112). A recent study showed that sperm increased expression of immune regulatory cues (i.e. IL-10, BMP4, and TGFβ) and reduced inflammatory cytokine and chemokine expression in the oviduct indicating that sperm are capable of regulating the oviduct immune response (113) but the signaling pathways responsible for sperm-induced immune regulation remain unknown.

3.3.8. Sperm release from the reservoir.

Compared to sperm binding, there has been less study of sperm release from the isthmus. First, there is an unresolved question of whether sperm are gradually and constantly released from the reservoir or if, instead, there is a signal for sperm release that causes many of the stored sperm to be released in a synchronized manner. One potential activator of sperm release is sperm capacitation. When capacitated sperm are added to oviduct cells or immobilized versions of the two porcine sperm binding glycan motifs, significantly fewer capacitated sperm bind to either the cells or glycans (95, 96, 114) so when capacitated sperm are released, it is unlikely that they would re-bind to the epithelium. This may be due to the loss or modification of specific proteins originating from male accessory glands that bind to sperm at ejaculation and then bind sperm to the isthmus (115, 116). However, binding to oviduct cells and glycans seems to delay some aspects of capacitation including Ca²⁺ influx, as discussed above (90, 91, 97). Another release trigger could be a burst of fluid flow due to contractions of the oviduct smooth muscle that could induce localized release (117). Finally, mathematical modeling of the additional force generated by sperm once their motility becomes hyperactivated suggests that hyperactivation might directly induce sperm release even if capacitation has not been completed (118). This was formally tested with porcine sperm. Various pharmacological approaches to induce hyperactivation promoted sperm release from oviduct cells and immobilized glycans before induction of capacitation (119). Progesterone also induced hyperactivated motility and sperm release in vitro and may be a physiological trigger of sperm detachment from the reservoir (118, 119). The source of progesterone has been debated. One potential source is the cumulus-oocyte complex (120, 121), which may contribute to the release of sperm from the ampulla (see below). The distance between the cumulus-oocyte complex and the isthmus as well as the fluid flow rate in the opposing direction presents a challenge for cumulus-oocyte products to act on sperm in the isthmus. Another potential source of progesterone is the ovary. Progesterone produced by the ovulated follicle can be carried by local transfer from the ovarian vein to the utero-tubal artery where it could be carried to and act on the isthmus (122–124). There may be multiple ways that sperm can be released to advance in the oviduct to provide a supply of sperm to the ovulated oocyte. Particularly in litter-bearing species in which ovulation is spread over longer periods, heterogeneous release may be advantageous for fertilization. Finally, the release of sperm bound to the ampulla appears to have different requirements than release from the isthmus, suggesting that they may be differentially controlled (125).

3.3.9. Regulation of sperm movement through oviduct fluid.

There is controversy about how sperm move through the oviduct. Sperm could either be moved by the fluid in which they are suspended or they could be attracted to oocytes by some type of taxis. Movies of fluorescently-labeled sperm in the oviduct showed some sperm attached to the isthmic epithelium, others suspended in oviduct fluid, and others “surfing” in oviduct fluid moving back and forth in the oviduct (10). The back-and-forth fluid movement was strongest in the isthmus and was significantly reduced in the ampulla (10). In addition, as sperm move into the ampulla where the space between the folds is smaller than the isthmus (Figure 2B), sperm change their motility so that they swim slower, spend more time at the epithelium interface, and have a lower beating amplitude (126). This is only one example of how moving into the ampulla affects sperm behavior. The vigorous fluid and sperm movement in the isthmus and contrasting slower movement in the ampulla have implications for sperm rheotaxis, chemotaxis, and thermotaxis.

3.3.10. Rheotaxis.

It has been known for many years that sperm orient themselves to navigate upstream at fluid flow rates that are moderate, a phenomenon called rheotaxis. Rheotaxis has been proposed as a way to provide directionality to sperm movement, primarily in the oviduct (127). Sperm can change their rotation and reorient themselves to a change in fluid flow direction (127), an accomplishment that was proposed to be dependent on the major Ca²⁺ channel in sperm, a multisubunit channel named CatSper (128, 129). More recent studies of sperm that are deficient in CatSper have shown that the rotation of sperm that promotes rheotaxis could be accomplished by sperm deficient in CatSper and flagellar Ca²⁺ signaling (130). Rheotaxis may be explained by more passive, non-signaling biomechanical, and hydrodynamic properties of sperm (131).

Because rheotaxis orients sperm in an upstream direction and the net overall fluid flow rate in the lumen of the oviduct at estrus is in the direction of the ovary, there is some question about the importance of rheotaxis in sperm movement in the oviduct (10, 80). But there may be specific roles for rheotaxis. The flow rate in the ampulla is much slower than in the isthmus (10) and there may be microcurrents, for example, controlled by ciliary beating along the walls of the ampulla, in which rheotaxis may operate.

3.3.11. Chemotaxis.

Sperm chemotaxis to a variety of targets has been demonstrated in vitro by many groups. But their role in vivo is still uncertain. The back-and-forth fluid movement in the isthmus (10) is expected to prevent the formation of chemotactic gradients, and the net fluid flow at estrus towards the ovary makes them unnecessary (80). On the other hand, chemotaxis may be important in the ampulla, where there is less fluid movement, at least in rodents (10). Cumulus cells secrete progesterone (120, 121) which has sperm chemoattractant activity (132, 133). But it is not clear whether cumulus cells produce adequate progesterone to form a chemoattractant gradient and attract sperm in the ampulla.

3.3.12. Thermotaxis.

Sperm from many species are thermotactically active (134); that is, sperm change their direction of motility according to a temperature gradient over a range in temperatures in vitro (i.e. 29–41°C) (135–137). This results in sperm accumulation at the location of higher temperatures (134, 137). Measurements of the temperature of pig and rabbit oviducts revealed that the end of the oviduct near the ovary was 1–2°C warmer than the UTJ, leading to the proposal that thermotaxis would aid in the movement of sperm to the ampulla (138).

It was proposed that thermotaxis acts over the entire oviduct, a greater distance than chemotaxis (134) due to the stability of the differences in temperature and the sensitivity of sperm to small differences in temperature. Rather than differences in temperature changing sperm metabolism, temperature information affects intracellular Ca²⁺ signals, the soluble adenylate cyclase/cAMP/PKC pathway, and protein phosphorylation (139–141). The temperature sensors in sperm are under investigation and may be opsins, a subset of G protein-coupled receptors, or ThermoTRP, a subset of the cation channels from the TRP superfamily (134). Transient receptor potential channels (dubbed TRPs) are multifunctional signaling molecules that include ThermoTRPs which are activated by temperature (142–146). The vanilloid-sensitive TRPs (TRPV) are the ThermoTRPs that have been studied most extensively in sperm thermotaxis in vitro (134). Although it seems clear that temperature influences sperm motility patterns and overall sperm movement, the in vivo role of thermotaxis is not completely clear. But thermotaxis may provide an additional mechanism whereby sperm move through the complex folds of the ampulla to oocytes.

4. NEW APPROACHES TO THE STUDY OF SPERM IN THE FEMALE TRACT

Sperm transport in the female tract is complex due to the many factors that must be integrated to understand the process. A recent and intriguing report used mathematical modeling to explain how sperm respond to the challenges they face, such as differing fluid viscosity, fluid microchannels, chemotaxis, rheotaxis, thermotaxis, epithelium complexity, and changes in adhesion molecules along the pathway (147). Sperm movement from the bovine vagina to oviducts was modeled, including things such as genital tract geometry, sperm motion, rheotaxis, and thigmotaxis (contact with a wall directing sperm movement). The computational model that was developed emphasized the importance of sperm velocity and directional stability (linearity) as well as sperm-fluid interactions and swimming along a wall to provide directionality to the ability of sperm to reach the oviduct (147). The use of computational modeling to make inferences about the complexity of sperm transport will likely provide new insights into questions, such as why so many sperm are required (1) and how sperm selection is accomplished by the female tract. Mimicking the selection applied by the female tract to select sperm for assisted reproduction techniques might also improve the success of these techniques (148).

5. CONCLUSIONS

Although there appears to be some selection for the ability of sperm to swim linearly with high velocity along the walls of the female reproductive tract in viscoelastic fluid, selection occurs primarily at the cervix and UTJ following semen deposition in the vagina but chiefly in the UTJ when semen is deposited in the uterus. Among the sperm that meet the selection criteria, those that are randomly moved to the isthmus most quickly, avoiding uterine NETosis, seem to then become candidates for the fertilizing sperm. And the sperm that are released from the isthmus and carried to the ampulla so that their entry into the ampulla coincides with the arrival of a mature oocyte could become fertilizing sperm. Superimposed on this selection is the random chance that sperm will be among the group that advances quickly to the isthmus and is released from storage in synchrony with the arrival of the cumulus-oocyte complex. The development of more sophisticated imaging technologies and computational models to integrate the fundamental information learned offer promise to refine our understanding of how sperm and oocytes meet, and how fertilization occurs in such a complex environment.