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provided by Elsevier - Publisher Connector Current Biology 23, 443–452, March 18, 2013 ª2013 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2013.02.007 Article Guides Mammalian Sperm

Kiyoshi Miki1 and David E. Clapham1,2,* approach the egg [5, 6]. Another hypothesis, thermotaxy, is 1Howard Hughes Medical Institute, Department of Cardiology, based on ovulation-dependent small temperature gradients Manton Center for Orphan Disease Research, Boston between the isthmus and ampulla [7]. Children’s Hospital, 320 Longwood Avenue, Enders 1309, Rheotaxis, observed in many aquatic species, is the orienta- Boston, MA 02115, USA tion of cells and organisms within a gradient of fluid flow. Posi- 2Department of Neurobiology, Harvard Medical School, tive rheotaxis, the tendency of a cell to swim against the flow, Boston, MA 02115, USA was first reported for spermatozoa over 100 years ago [8]. While ciliary current toward the distal oviduct was observed in turtle, pigeon, and rabbit, central lumen counterflow pre- Summary vented a consensus about the physiological relevance of rheo- , and it was concluded to be a laboratory artifact [9]. In Background: In sea urchins, spermatozoan is altered addition to cilia-directed flow, muscle contraction, tortuous by chemotactic peptides, giving rise to the assumption that geometry, the propensity of sperm to stick to surfaces, and mammalian eggs also emit chemotactic agents that guide high-viscosity fluids were recognized as factors complicating spermatozoa through the female reproductive tract to the sperm transport. Rothschild [10] noted that the sperm head mature oocyte. Mammalian spermatozoa indeed undergo is attracted to surfaces (the wall effect), and suggested that complex adaptations within the female (the process of capac- the ‘‘lighter’’ tail is subjected to more force in velocity gradi- itation) that are initiated by agents ranging from pH to proges- ents, resulting in the tail being dragged downstream with the terone, but these factors are not necessarily taxic. Currently, head pointed upstream [11]. More recent considerations , thermotaxis, and rheotaxis have not been defini- explain the wall effect as a consequence of hydrodynamics tively established in mammals. in tubes [12]. Results: Here, we show that positive rheotaxis, the ability of Fluid viscosity in the female reproductive tract varies, with organisms to orient and swim against the flow of surrounding the highest being in the cervical mucus (200 to 680 cP in hu- fluid, is a major taxic factor for mouse and human sperm. This mans [13]) and the oviductal isthmus [14]. Oviductal viscosity flow is generated within 4 hr of sexual stimulation and coitus in varies during the estrus cycle. Around ovulation, ciliated cells female mice; prolactin-triggered oviductal fluid secretion in the isthmus epithelium are covered by a dense, tenacious clears the oviduct of debris, lowers viscosity, and generates mucus that disappears after ovulation [14]; estrogen-regulated the stream that guides sperm migration in the oviduct. Rheo- secretion of oviductal fluid increases 2- to 3-fold at estrus [15]. taxic movement is demonstrated in capacitated and uncapaci- Fluid viscosity also affects sperm movement—as viscosity tated spermatozoa in low- and high-viscosity media. Finally, increases, sperm flagella propagation velocity, wave ampli- we show that a unique sperm motion, which we quantify using tude, and energy expenditure decrease [16]. Sperm angular the sperm head’s rolling rate, reflects sperm rotation that speed (rotation around the long axis of sperm) also decreases generates essential force for positioning the sperm in the at higher viscosities, resulting in more planar movement [17]. stream. Rotation requires CatSper channels, presumably by In the complex process of capacitation (that is, the changes enabling Ca2+ influx. that sperm undergo in the female reproductive tract that Conclusions: We propose that rheotaxis is a major determi- enable them to fertilize the egg), sperm develop hyperacti- nant of sperm guidance over long distances in the mammalian vated motility. Hyperactivation is characterized by higher- female reproductive tract. Coitus induces fluid flow to guide amplitude, asymmetric waveforms of reduced beat frequency sperm in the oviduct. Sperm rheotaxis requires rotational [18], which enables high flagellar swimming speeds in viscous motion during CatSper channel-dependent hyperactivated solutions [19]. motility. We have reinvestigated sperm taxis in vitro and in vivo using new tools that enable control of hyperactivated motility and Introduction enable the visualization of sperm movement in vivo. Here, we show that sexual intercourse in mice induces robust secretion Marine invertebrate spermatozoa, such as those of sea urchin, and fluid flow within the oviduct. Sperm are directed by, are guided by chemotaxis; egg-secreted peptides induce and swim against, this flow (positive rheotaxis). Importantly, changes in the arc of spermatozoan swimming paths that axial rotation requires CatSper channels for reorientation of increase fertilization success [1]. In mammals, the route to sperm in tangential flow. We conclude that rheotaxis is a major the egg is more restricted, but it is widely assumed that folli- influence in the long distance guidance of mammalian sperm cular fluid contains chemoattractants [2]. Prompted by reports to the egg. of the presence of a large number of putative odorant G protein-coupled receptors in spermatozoa, the synthetic odor- Results ants bourgeonal and lyral were proposed to mimic unknown native chemoattractants of human or mouse sperm, respec- Coitus Induces Fluid Flow from Oviduct to Uterus tively [3, 4]. However, because muscle contraction and ciliary Oviductal epithelia actively secrete fluid into the oviductal currents would disrupt gradients over long distances, chemo- lumen, as first reported in rabbits during estrus [20]. Uterine tactic guidance would likely be relevant only as sperm fluid is most abundant (91 6 26 ml) on the day of coitus [21]; however the origin of fluid and the mechanism of its production has not been well documented. We analyzed the accumulation *Correspondence: [email protected] of oviductal fluid in the uterus in mice. Although the uterus Current Biology Vol 23 No 6 444

A C Figure 1. Increased Fluid Production and Oviductal (i) (ii) (iii) (iv) (i) (ii) (iii) Flow after Mating (A) The mouse uterus swells after mating. No fluid was recovered from the uterus at ovulation (i). Fluid from uteri removed immediately after mating increased by 37 mg, consistent with the weight (volume) of sperm fluid (ii). Fluid volumes increased markedly in uteri 8 or 12 hr after mating (120 or B 100 mg, respectively) (iii and iv). Scale bar repre- sents 1 cm. (B) Accumulated uterine fluid was collected at the D times indicated after mating. Fluid weights (volumes) from mated (red) and unmated (blue) females are plotted. (C) Uterine fluid was collected from females 20 min (i), 4 hr (ii), or 8 hr (iii) after mating, and the fluids were centrifuged. (D) Fluid flow in the oviduct (ampulla) 4 hr after mating. Migration of globular cells and debris was traced over 3 s; the closed circle marks the endpoint. The central diagonal structure is a mucosal fold in the Recovered fluid (mg) oviductal wall. Oviductal isthmus is seen at bottom right. Scale bar represents 50 mm. See Movie S1. (E) Average globular cell migration speed = 18.0 6  Hours after mating 1.6 mm/s at 22 C. (F) administration before mating E F p = 0.0007 G inhibits fluid secretion measured 8 hr after mating. p = 0.008 (G) DsRed2-expressing sperm counts in the ampulla in vitro and ex vivo. Ex vivo: a portion of ampulla was dissected from females 4–5 hr after mating, and DsRed2-expressing sperm were counted. Oviducts were removed and ligated, or left unligated, near the UTJ 60–90 min after mating and then incubated at 37C for 2–3 hr. The number of sperm reaching the ampulla in oviducts without an ovary averaged 31 6 6. The increase in sperm number over the in vivo situation may result from manipulation or a change in hormone status perhaps detaching some sperm # sperm / ampulla Flow speed (µm/s) from the isthmal crypts and facilitating sperm migra- Recovered fluid (mg) tion. All graphs: mean 6 SEM (black bars) and Vehicle BrCr in Unligated Ligated median with interquartile ranges (green boxes). vivo ex vivo noticeably swells at estrus, no appreciable fluid was recovered reducing the attachment of cells to the wall of the oviduct from ovulated females at any time point up to 24 hr after estrus (Figure 1D; Movie S1B). Ampullar flow, measured 3–4 hr after (Figure 1B). When females were mated with males, we coitus, was 18.0 6 1.6 mm/s (Figure 1E). In summary, coitus observed a slight swelling of the uterus 20 min after mating induces a dramatic increase in oviductal fluid secretion that and were able to collect 22 6 4.8 mg of fluid (primarily ejacu- lowers viscosity and clears debris from the oviduct. We then lated semen; Figures 1Aii and 1Ci). Moreover, fluid in the uterus asked what factors might mediate this increased oviductal increased over time to a peak of 87 6 13.9 mg at 8 hr (Figure 1B; secretion and flow. the rate of secretion appeared to be highest within the first Genital stimulation and coitus evoke complex neurological 4 hr after mating). This increase in secretion above baseline responses. To simplify our task of determining factors that at estrus required coitus; unmated control mice did not exhibit might increase oviductal flow, we considered known secreted a significant increase in uterine fluid. Because in vitro culture hormones that were (1) elevated in serum after sexual stimula- showed that the uterine accumulation included oviduct- tion or coitus, (2) known to effect fluid secretion across released cell debris and globular cells (see Figure S1A epithelia surfaces, or (3) known to alter fertilization success available online), we surmised that some portion of this fluid in mice in which the gene had been deleted. These criteria in vivo originates from oviductal fluid. narrowed our candidate hormones to prolactin, , Oviductal fluid pH remains fairly constant during the follic- and . Prolactin is released from the anterior pitui- ular phase of the estrus cycle. During ovulation in rhesus tary in response to sexual arousal circuitry that releases dopa- monkeys, bicarbonate secretion into the oviduct increases mine onto neuroendocrine cells. Prolactin, in turn, counters its pH from 7.1–7.3 to 7.5–7.8 [22]. Mouse uterine fluid recov- dopamine’s mediation of sexual arousal and reduces sexual ered within 4 hr after coitus was a highly viscous colloid of receptivity [23]. In humans, plasma prolactin concentrations seminal fluid, cell debris, and globular cells, but the fluid’s are increased for >1 hr following orgasm induced by masturba- viscosity decreased over time (Table S1). Beginning a few tion or sexual intercourse [24]. Female mice lacking the hours after coitus, significant secretion of bicarbonate-rich prolactin receptor gene have multiple reproductive defects, oviductal fluid increased the pH to w8.3, reduced mucous including reduced fertilization rate [25]. Finally, prolactin also viscosity, and washed out viscous cellular debris (Figure 1C; has a plethora of effects on secretory epithelia [26]. To test Movie S1A). Cilia sweep fluid and the egg toward the uterus, whether prolactin could mediate oviductal secretion, we Sperm Rheotaxis 445

Figure 2. Sperm Rheotaxis In Vitro A 33.5䉝 (surface) B G (A) Diagram of convective flow pattern observed in warmed media. (B) Laminar flow (1.5 ml/hr) from a rectangular capillary at 37C. 37 (bottom) (C–F) Trajectories of mouse (in C, 3 s; in D, 4 s), and human (in E and F, 5 s) sperm in flow, No flow C D analyzed by CASA. Scale bars represent 200 mm m H p = 10-6 (C and D) and 100 m (E and F). (G) Positive rheotaxis is defined here as sperm n = 5 flow movement against fluid flow within 622.5 of (288) p = 0.0002 the forward vector of sperm movement. Mouse n = 3 (H) Rates of rheotaxis for mouse and human (270) sperm (mean 6 SD) in five and three independent experiments, respectively. Total sperm count is E No flow F shown in parentheses. See Movies S3 and S4. n = 5 n = 3 (467) (240) flow

Human Mouse Human and no fertility phenotypes in TRPC5, V1, V3, A1, or M8 knockout mice [29–33]. Furthermore, we failed to detect any of these TRP currents in voltage-clamped mouse spermatozoa. Most tellingly, administered the dopaminergic receptor agonist bromocrip- we noticed that temperature gradients create convection tine (BrCr) 11 hr before coitus to inhibit prolactin secretion currents that influence the direction in which sperm swim. As from the anterior pituitary. BrCr treatment reduced mouse the chamber warms the medium from the bottom surface, fluid uterine fluid accumulation (Figure 1F). In contrast, administra- flows in well-recognized convection patterns, reaching tion of the VR2 receptor antagonists oxytocin (atosiban) or a steady state in which fluid cycles from bottom to top and vasopressin () did not suppress oviductal secretion. then down the sides of the chamber (Figure 2A). Sperm consis- We conclude that prolactin initiates the increased oviductal tently swam against this flow (positive rheotaxis), whether fluid flow to the uterus after coition. We next asked whether along the top liquid surface (in the direction of increasing oviductal flow affected sperm movement. temperature) or bottom (in the direction of decreasing temper- ature) of the dish (Movies S2A and S2B). Solutions flowing Sperm Migration in the Oviduct through a pipette into a dish at constant temperature initiated Ejaculated sperm migrate through viscous fluid to bind to identical positive rheotaxis (Movie S2C). To quantify rheotaxis, epithelial cilia in the oviductal isthmus [6]. We tested whether we defined rheotactic movement as movement within 622.5 sperm then detached from the cilia and swim to the ampulla of the forward vector of sperm movement (swimming directly after the mucus fluid had been cleared. Taking advantage of upstream; small particles defined the local vector of flow; Fig- genetically modified sperm, in which enhanced green fluores- ure 2G). Providing the baseline in static media, we observe cent protein (EGFP) is expressed in the sperm acrosome and 12.5% of sperm in the field swim in the direction specified by DsRed2 fluorescent protein is expressed in sperm mitochon- the 45 arc centered along the sperm longitudinal axis. Without dria [27](Figure S1C), we found that 9 6 1 sperm reached flow, mouse and human sperm swam in random directions the ampulla 4–5 hr after mating (Figure 1G; in vivo). In order (Figure 2H). to test whether rheotaxis was functional ex vivo, oviducts were dissected from mice 60–90 min after mating. By this Rheotaxis of Uncapacitated and Capacitated Sperm in time, sperm had reached the isthmus, and in the next 2–3 hr, Low-Viscosity Media they migrated from the isthmus to the ampulla. Importantly, Because sperm capacitation can vary with respect to position sperm in the isthmus migrated successfully to the ampulla and time in the female reproductive tract [18], we evaluated ex vivo in the absence of an ovary, a source of potential che- sperm rheotaxis in both uncapacitated and capacitated moattractants (Figure 1G). However, sperm transport was mouse sperm. Ejaculated sperm swim to the oviductal isthmus limited when oviducts were ligated near the uterotubal junction through the UTJ within 1–2 hr, but most require several more (UTJ) to block fluid flow (Figure S1B), as assessed by observa- hours to reach the ampulla [6]. When uncapacitated mouse tion of DsRed2-tagged sperm upstream of the ligation site and or human sperm were added to the convective flow chamber, by the lack of discharge of cell debris and globular cells. Sperm 68% and 51% of the actively motile sperm exhibited rheotaxis, motility in the blocked oviduct appeared normal, but there respectively (Figures 2C–2F and 2H; Movies S3 and S4). To re- were w3-fold fewer sperm in the ampulla of the ligated move confounding results due to potential thermotaxis, we oviducts (11 6 2), indicating that fluid flow is a factor in optimal repeated these experiments in isothermal fields of flowing sperm transport ex vivo. media. Uncapacitated sperm were placed in shear flow (Rey- nolds number [Re] = 0.002) via a 1 3 0.05 mm (w 3 h) capillary Spermatozoan Rheotaxis tube (Figure 2B). As expected, spermatozoa swam against the The suggestion by several groups that transient receptor direction of flow (positive rheotaxis; Movie S2C), whereas in potential (TRP) channels are present in spermatozoa and controls (no flow) sperm movement was randomly directed might mediate sperm thermotaxis [28] motivated our initial (Movie S2A). studies to examine sperm movement in a thermal gradient. Laminar flow in a chamber varies due to surface drag; However, we found no effects on sperm motility or fertilization velocity is highest in the center of the stream and falls to Current Biology Vol 23 No 6 446

Outward flow Figure 3. Rheotaxis of Mouse Sperm in Low- and A High-Viscosity Media exiting (A) Capacitated (Cap) or uncapacitated (Uncap) sperm were suspended in medium with (high- Loading viscosity) or without (low-viscosity) 0.3% (w/v) Inward flow methylcellulose. Sperm were loaded into a capil- lary (at 22C), and positive (outward flow; w15 nl/ inner diameter: 80 µm exiting min) or negative (inward flow; w15 nl/min) pres- sure was applied (at 37C). The most immotile B C and sluggish sperm were present in the central Low viscosity High viscosity stream (w50 mm/s flow rate), whereas most of Loading (804) (814) the motile sperm swam near the wall where the flow rate was lowest. a (509) c (633) FlowOUT (B and C) Sperm were categorized as motile,

Uncap sluggish, or immotile. Sperm movement near Flow (587) a (531) c Exiting IN the end of the capillary was observed under Loading (877) (843) outward (FlowOUT) or inward (FlowIN) flow in the low- (B) or high-viscosity (C) medium (mean 6 b (825) d (697) FlowOUT SD, n = 5; total sperm counts are shown in Cap parentheses). Statistical significance was com- Flow (616) b (544) d Exiting IN pared between the number of motile sperm

in FlowOUT and FlowIN. p values of each experi- Sperm (%) Sperm (%) ment (compare error bars marked as a, b, c, 26 Motile Sluggish Immotile and d) were smaller than 10 . See Movies S5, S6, and S8.

a minimum next to the walls [34]. The flow gradient was sperm and eventually to the uterus. Thus, oviductal secretion quantified by measuring 1 mm spherical beads placed in the and flow clear the oviduct and increase pH to induce hyperac- fluid as markers. Velocity profiles in fluid chambers are para- tivation; rheotaxis selects for motile sperm and ensures that bolic and approach linearity only near a small portion of their eggs are fertilized while still in the oviduct. As proposed by profile near a boundary. By tracking beads, we found that Suarez and colleagues, slow release from reservoirs of quies- flow speed increased in the range of 0–300 mm from the cent sperm or sperm attached to epithelial or ciliary surfaces surface. Bead velocity in the range of 0–80 mm could be may provide a steady supply of sperm as this process is approximated as w1.45x, where x is the distance in microns repeated over time [6]. from the surface (least-squares r2 = 0.98). The swimming tracks of added sperm appeared as ‘‘threads’’ (similar to those Sperm Movement in High-Viscosity Media in Figures 2D and 2F), swimming w9 mm from the surface (flow During estrus, high-viscosity mucus is an obstacle to sperm velocity = 14.5 mm/s). In this low-viscosity fluid, capacitated progress through the isthmus lumen. Assuming, as a first mouse sperm that swim into the higher velocity of the central approximation, that the fluid is Newtonian, we measured the stream are swept downstream. Sperm swim near the surface viscosity of secreted and accumulated lumenal fluid in the where flow rate is slow, and those that stray away from the uterus, in which oviductal mucus, cell debris, and ejaculated surface are swept away by the faster, more central flow under semen were present. The viscosities of this fluid averaged these conditions. 81 6 73 cP compared to the viscosity of clear fluid superna- The lumen of the mouse oviductal isthmus consists of tants (2.4 6 0.9 cP; Table S1). One caveat however, is that a narrow central channel surrounded by pockets formed by the fluid is thixotropic (viscosity decreases with shear) and deep transverse mucosal folds. Sperm in the mucosal folds thus likely non-Newtonian. Nevertheless, we estimate that can access the central channel to migrate in the oviduct. To the viscosity of lumenal fluid decreases to as low as 2–3 cP simulate the narrow central channel, sperm were capacitated upstream of the congestion of mucus and cell debris in the for 2–3 hr in human tubal fluid (HTF) medium (cooled to 22C isthmus. Therefore, we used media containing 0.3% methyl- to limit sperm motility) and loaded into the capillary (Figure 3A; cellulose (MC) (6.7 cP; Table S1) to mimic the environment of Movie S5A). The capillary was then transferred to a 37C the oviductal lumen. As seen in Figure 4A and Movie S7A, un- chamber where sperm resumed more active motility. Under capacitated mouse sperm swim in circles in viscous medium outward-directed flow (FlowOUT; w15 nl/min or w50 mm/s), regardless of priming by bicarbonate ion in HTF medium sperm motility was recorded and sperm that had been swept (w3 min), which increases flagellar beat frequency within out the end of the capillary counted. Whereas 44% of the 30 s [35]. The majority of uncapacitated sperm exhibit planar sperm loaded into the tube were immotile, 78% of sperm swimming (Figure 4A), with one side of the head facing the swept from the tube were immotile (Figure 3B; Movie S5B). surface. This swimming behavior may increase the chance Conversely, when sperm were slowly sucked into the for uncapacitated sperm to stick to oviductal epithelial cells pipette (inward flow; FlowIN; w15 nl/min or w50 mm/s; 39% more frequently, a delay that might promote acquisition of motile), 93% of sperm were active and moving against fluid the capacitated state [36]. flow, leaving immotile and some sluggish sperm behind (Fig- ure 3B; Movie S6). Thus, gentle flow concentrates, or selects, Sperm Rotate after Capacitation in Viscous Media viable sperm. Uncapacitated sperm showed essentially the When sperm are observed under phase-contrast microscopy, same results (Figure 3B). In short, sperm swim upstream in reflection intensity varies sinusoidally as the sperm head a capillary mimicking flow rates in the oviductal lumen, a direc- rotates, with the highest intensity corresponding to the head tion that also moves the oocyte toward the incoming in the position vertical to the surface [13](Figure 4C). We Sperm Rheotaxis 447

Figure 4. Sperm Rotation during Capacitation A D 250 (A and B) Sperm trajectories in viscous solu- tion: sperm were incubated with 0.3% (w/v) methylcellulose-containing HEPES-buffered HTF medium for w3 min (A: uncapacitated) or 2 hr (B: capacitated). Scale bar represents 200 mm. See Movie S7. 150 (C) The head of the spermatozoa is dark on uncapacitated phase-contrast images due to its relatively flat Brightness (a.u.) 0 0.5 1.0 structure (upper panels). Sperm head light reflec- Seconds tance increases on phase-contrast images when the sperm heads orient vertically to the surface E (lower panels). 250 B (D and E) Representative results of the sperm head-rolling (rotation) analysis. Uncapacitated (D) or capacitated (E) sperm were incubated in HTF medium for 3 hr and analyzed (a.u. indicates arbitrary units). 150 (F) Increase of rotation rate during capacitation capacitated (6SD; w70 sperm for each time point). Brightness (a.u.) 0 0.5 1.0 Seconds F 8 fertilization site faster than uncapaci- tated sperm in high-viscosity solution. C 6 Increased Rotation in Turning As noted above, sperm often swim close Flat 4 to surfaces due to the hydrodynamic wall effect [10]. We observed that sperm Vertical 2 eventually leave the surface, are exposed to faster flow and pushed Rotation rate (Hz) downstream, but then they return to Incubation (min) the surface and again orient against 0 50 100 150 200 fluid flow (Figure 5A; Movie S9). We noticed rapid sperm rotation (3.8 6 0.2 Hz; 6SEM) when the sperm turns in plotted sperm head reflection intensity as a function of time response to fluid flow at a position perpendicular to the direc- and call it ‘‘rotation frequency,’’ because it reflects the spin- tion of fluid flow (11 out of 14 analyzed sperm; Figure 5B), ning of the sperm head and tail around a longitudinal axis. followed by a return to normal rotation frequency (3.0 6 Rotation frequency was 1.7 6 0.1 (6SD)-fold higher in capaci- 0.2 Hz; 6SEM). Time-course analysis of turning sperm re- tated (Figures 4E and 4F) than uncapacitated (Figures 4D vealed a peak rotation rate of 7.1 Hz during turning, compared and 4F) sperm. During capacitation, the linear progress of to 4.5 Hz prior to turning (Figure 5C, red). In contrast, the rota- the movement is increased by rotation (Figure 4B; Movie tion frequency of sperm in steady-state positive rheotaxic S7B). In flowing high viscosity media, uncapacitated sperm movement was constant during steady flow and indistin- swim in a corkscrew path, and the head does not rotate guishable from sperm in ‘‘no-flow’’ conditions (Figures 5C when closest to the wall (Movie S8A). This motion may be and 5D). To further examine the propensity of sperm to turn boundary-driven (thigmotaxic) rather than rheotaxic. Sperm into a stream we examined sperm at the confluence of two rotation after capacitation may enhance detachment from flow streams 45 apart (Figure 5E). In these situations, sperm surfaces. Once in the stream, linear progress is enhanced; rotation frequency significantly increased and resulted in the head rotates and the sperm progression pattern oscillates sperm being directed into the stronger stream. Since inertia around a motion-oriented parallel to the flow (Movie S8B). is negligible in the microscopic range [12] but governed by Linear velocity (migration speed toward the capillary end) of overdamped, zero Reynolds number dynamics, the system uncapacitated and capacitated sperm was 36 6 6 and 54 6 will be governed not by momentum, but rather by relative 8 mm/s (mean 6 SD, n = 11), respectively, under comparable forces. Our conclusion is that sperm increase their rotation flow conditions in viscous media (Movie S8). In summary, rate when they encounter tangential (side) forces. sperm rotation after capacitation enables sperm to swim into the main fluid stream (Figure 3C; Movie S8B). At the Mechanisms: Tail Rotation Is Required for Rheotactic boundary (chamber floor), the fluid velocity approaches zero, Movement but as the sperm head moves up into the fluid, sperm experi- Orientation in a flowing stream has been described for ence a difference in flow velocity that rotates the cell. This mammalian sperm [11, 35] and bacteria [37]. Some mecha- previously unappreciated capacitation-induced movement is nistic models of this orientation assume stronger hydrody- an important aspect of motion, in addition to the increased namic drag on the head (sperm) or cell body (bacteria) than motility of sperm in viscous medium reported by Suarez the flagellum that orients the front of the cell toward the et al. [19]. Thus, the gain in forward progression in the surface, resulting in the tail being dragged downstream with oviductal lumen enables capacitated sperm to reach the the head and cell body pointed upstream [11, 37]. To separate Current Biology Vol 23 No 6 448

A Figure 5. Increased Sperm Rotation Rate in Fluid Flow (A) Representative uncapacitated spermatozoan turns in response to fluid flow. Images were re- corded at 0.5 s intervals; black arrows mark a turning sperm. Flow direction, in all panels, is indicated by the green arrow. See Movie S9. (B and C) Rotation rate of individual turning sperm; average of two 1 s periods plotted at 0.5 s intervals (some points overlap and are C shown as single dots) (B) or sequentially over time (C). Red line in (C) indicates a turning sperm; other lines indicate sperm swimming in a straight B Perpendicular turning sperm line against fluid flow. (D and E) Conditions that change sperm rotation 1 s 1 s rate. Uncapacitated sperm were incubated at 37C, and the rate of rotation measured in no 0.5 s flow, steady flow (14 mm/s), or convergent flow } (shown in E; 6SEM, n = 35). non-turning sperm Rotation rate (Hz)

fluid flow; their trajectories were similar Seconds 1 2 to those of mouse sperm lacking CatS- per channels or uncapacitated sperm in D p = 0.0003 E high-viscosity medium (Figure 6B; p = 0.0004 convergent Movie S12A). Regardless of fluid flow, flow these sea urchin sperm swam in circular planes (Movie S12B), consistent with the behavior of sperm from marine Rotation rate (Hz) invertebrates that are not guided by marine currents but swim in concentric p = 0.0001 circles until encountering chemoattrac- tants that turn and widen their arcs of pipette circular motion [39]. Rotation rate (Hz) Prior studies show the sperm Turn Linear convection flagellum oscillates in a single plane when tethered at its head on a glass coverslip [35]. In order to better under- stand the motion of sperm in three dimensions during free swimming, we recorded a sperm flagellum every 50 ms and aligned these waveforms by horizontal translocation of the the differential influence of drag on head and tail, we examined swimming direction axis (Figure 7A). During free swimming, swimming headless sperm, which represent a minute fraction flagella crossed the x axis at w20% of their entire length as (<0.1%) of sperm from normal mice. Of these, 82% (14 out of measured from the head (Figure 7A, right). This point is the 17) swim against fluid flow (Movie S10), indicating that the minimum of flagellar excursion (Figure 7, arrowhead) and head does not act as an aft rudder and is not required for moves along the x axis with minimal y deviation or yaw into normal orientation—in other words, the rotating tail alone the plane (z axis). If sperm do not rotate, flagellar trajectories can exhibit positive rheotaxis. trace a triangular plane, resulting in minimal rheotaxis. Sperm lacking any one of five CatSper subunits (CatSpers1–4, Sperm rotation produces trajectories that trace a symmetric CatSperd) exhibit identical phenotypes [38], fail to develop cone-like path (Figure 7B), consistent with previous observa- functional CatSper Ca2+ currents, and lack hyperactivated tions of human spermatozoa that trace out a cone-shaped motility. Uncapacitated sperm lacking ICatSper usually helical pattern and have an extended elliptical cross section swam in a counterclockwise circular plane on the bottom when their heads are immobilized by attachment to a micropi- surface, as observed from the top (Figure 6A; Movie S11A). pette, but allow the tail to exhibit flexible three-dimensional This swimming behavior did not change regardless of fluid movement [40]. Tangential forces on the tail anterior to the flow (Figure 6A; Movies S11B and S11C), indicating that the minimum of flagellar excursion will produce a clockwise force CatSper channel was required for effective rheotactic (as seen from above), whereas those on the posterior tail responses to fluid flow. Presumably, calcium influx through produce a counterclockwise force. The posterior tail receives CatSper channels enhances sperm rotation, which changes stronger tangential force due to its higher proportion of the circular planar movement to rotational swimming. Thus, rota- flagellar length (w80%, presenting a greater cross section to tion is an essential sperm motion for proper rheotaxis, and the force of fluid flow), pointing the sperm upstream (Fig- CatSper channels contribute to this response. Interestingly, ure 7B). If the sperm were fixed at the point of minimal S. droebachiensis sea urchin sperm did not swim against excursion, this would be analogous to a weather vane that is Sperm Rheotaxis 449

A No flow CatSper KO A

B

B No flow Sea Urchin

flow

Figure 6. Without Rotation, Mouse and Sea Urchin Sperm Swim in Circles Trajectories of uncapacitated sperm from CatSper1 knockout mouse (A: 3 s) or sea urchin sperm (B: 2 s). Fluid flow was generated by convection or via pipette, and sperm trajectories were analyzed by CASA. Scale bar in (A) represents 200 mm; scale bar in (B) represents 50 mm. Green arrows show the direction of fluid flow. See Movies S11 and S12. Figure 7. Model for Sperm Rheotaxis (A) The flagellum of an unattached sperm swimming against fluid flow was divided by an axis of rotation into a small anterior and a larger traced every 50 ms and aligned by horizontal movement in the swimming posterior surface that receives stronger tangential force and direction (dotted line). Arrowhead indicates point of minimal excursion of flagellum (mef). thus guides the weather vane into the wind. A freely motile (B) Fluid flow (red arrows) reorients sperm (yellow arrows) into the flow to sperm in a homogeneous flow is not constrained to be reduce shear as the sperm rotate (orange arrow) and propel themselves stationary at any point and therefore is not subject to torque, upstream. Rotation maps out a three-dimensional cone shape in space, but it is subjected to differences in flow velocity along its which orients sperm consistently into the flow (positive rheotaxis). length.

Discussion Again, this does not mean that true thermotaxis or thermal effects on sperm motility are absent. We propose that rheotaxis, assisted by factors inducing hy- Fluid secretion in response to coitus has been relatively peractivated motility (alkaline pH and, in humans, proges- unstudied. We found that there was little fluid secretion into terone), is a major mechanism guiding sperm in mammalian the oviduct and uterus at ovulation in mice. In contrast, coitus female reproductive tracts. This rheotaxis is not related to induced a dramatic secretion of fluid that accumulated in the sensory mechanisms and guidance responses as it is in large uterus (pH w8.3), consistent with active extrusion of bicar- animals such as fish but a consequence of rotational motion bonate ion and water from the oviductal epithelium [42]. potentiated by calcium entry via CatSper channels. Three Interestingly, simulated coitus improves the success rate of arguments support sperm rheotaxis as a major guidance and artificial insemination in mice [43]. In humans, sexual stimula- selection mechanism. First, coitus triggers substantial fluid tion of females increases serum prolactin [24], and our initial secretion into the oviduct, thus increasing lumenal flow findings in mice suggest that this is mediated by bromocrip- directed by cilia and muscle contractions from ampulla to tine-sensitive dopaminergic pathway control of the anterior uterus. Second, positive rheotaxis of mature spermatozoa pituitary gland. Prolactin regulates water and electrolyte from mice and humans is observed in situ and in vitro. Finally, balance in many organs (e.g., kidneys, fish gills [26]), perhaps capacitating conditions and physiological fluid flow overcome via activation or regulation of the cystic fibrosis transmem- the wall effect and sperm adhesion—these factors accelerate brane conductance regulator (CFTR) [23, 42] or aquaporins sperm rotation that in high viscosity fluid evokes a change [44]. With increased oviductal flow, globular cells from the from planar circular swimming to directional swimming. In oviduct were also discharged into the uterus within 4 hr after contrast, the most popular hypothesis for taxis, chemotaxis, coitus. Apparently, ciliary motion and muscle contraction in lacks both bona fide chemoattractant candidates and cognate the isthmus pushes oviductal fluid to the uterus, in turn receptors in mammals. We cannot, however, exclude a role for washing away mucus and globular cells to clear the way for chemotaxis—if it occurs, it is most likely relevant over short sperm traffic. Sperm harbored in the lower isthmus then orient distances. Similarly, we conclude that our results cannot be and swim against this flow to reach the ampulla. Unlike explained by thermotaxis. In the study of Bahat et al. [41], the isthmus, countercurrents in the center of the ampullar only w5% of cells respond to temperature gradients, 8-fold lumen circulate fluid while ciliary currents push the fluid down- less than the percentage of sperm responding to rheotaxis in stream into the oviduct [9]. Due to the hydrodynamic wall our experimental system (w40% of the population; Figure 2). effect [10], sperm moving locally along ciliated epithelium Most importantly, we observed that sperm swam upstream may also swim against this ciliary current to reach the cumulus even though there was a descending temperature gradient. oophorus complex. Current Biology Vol 23 No 6 450

Sperm of marine invertebrates swim in a relatively unre- Observation of Oviductal Fluid Flow  stricted three-dimensional space, where the encounter of an Oviducts were removed 3–4 hr after mating and placed in a 37 C chamber egg may be fortuitous. Chemotaxis substantially improves containing M2 medium (2 ml; Specialty Media, Millipore). A cover glass was placed over the oviduct, and fluid flow was recorded through the the odds of reproduction of these species. In contrast, the oviductal wall (20 3 objective; CCD at 60 frames/s; 22C). reproductive tract of mammals is restricted to a quasi-one- dimensional tube with increasing pH (and progesterone in Ex Vivo Sperm Migration humans) initiating hyperactivated motility to free sperm from DsRed2-EGFP transgenic males [28] (B6D2F1-Tg; CAG/su9-DsRed2, Acr3- adherent surfaces and rheotaxis to guide the sperm toward EGFP RBGS002Osb) enabled clear sperm visualization. Sixty to ninety the oviduct. With up to 5 million mouse sperm deposited minutes after the end of the mating period, oviducts were removed and in the females after ejaculation, only w20 sperm cells reach ligated just after the UTJ (ovarian side). Oviducts were gently washed in HTF medium (Specialty Media, Millipore) to remove sperm attached to the the ampulla within several hours, similar to the success rate outside wall and then incubated (5% CO2) for 2–3 hr. A portion of the ampulla of other mammals [40]. Oviductal flow has two advan- was first cut with a fine scissors to prevent sperm from leaving the isthmus, tages—it guides sperm to the egg but also selects for strong, then the oviduct was stretched by trimming the mesosalpinx and placed on capacitated sperm. Mammalian sperm must penetrate a glass slide (14 each of ligated or unligated oviducts), and sperm inside mucus, the viscoelastic matrix of the cumulus oophorus, were counted under fluorescence microscopy. Samples in which there and the tough zona pellucida, requiring vigorous sperm for were obvious sperm aggregates on either side of the ampulla were removed from statistical analysis (two cases each; ligated and unligated oviducts). successful fertilization. Also, directional flow sweeps the egg down the oviduct, and sperm that swim against the flow Sperm Preparation preferentially fertilize the egg while still in the oviduct. This Mouse sperm were collected from the B6D2F1 male (Jackson Laboratories) provides time for the egg to develop for implantation. From cauda epididymis and incubated for 5 min to allow sperm to swim out. this viewpoint, rheotaxis is a logical guidance and selection Centrifugation at 900 g (5 s) removed debris and large sperm aggregates. 6 mechanism. Sperm were then incubated at a density of 2 3 10 /ml in M2 media for 30 min Our results suggest that positive rheotaxis is due to the or HTF medium for 2–3 hr (5% CO2), yielding uncapacitated or capacitated sperm, respectively. Cryopreserved human sperm were purchased from spiral rotation of the sperm tail that orients sperm upstream, Fairfax Cryobank. Actively motile human sperm were collected by the and as such, it is decidedly an active process. Calcium entry swim-down procedure. Frozen sperm (w3 3 107) in egg yolks were is required to trigger the increased amplitude of the distal thawed and washed by suspension in 5 ml HTF, followed by centrifugation tail excursion that increases tangential force. At the same at 500 g 3 5 min to obtain a sperm pellet. After two rinses, the pellets time, increasing angular speed stabilizes the sperm’s swim- were resuspended in 0.5 ml HTF. Half of the sperm suspension was overlaid with a 0.5 ml Percoll cushion (80% HTF, 20% Percoll, 0.1 M NaCl). ming orientation due to increased motive force against the  Sperm were incubated for 45 min in a 37 C/CO2 incubator and sperm surroundings. Because the hyperactivated flagellar wave- swimming-down into a Percoll cushion were recovered by centrifugation form is both asymmetric and of larger amplitude [18], the at 1,800 g 3 1 min. Sperm in the pellet were resuspended in 0.5 ml HTF flagellum receives larger tangential forces, especially in and incubated for 30–60 min. high-viscosity solution [6], which points the sperm into flow- Green sea urchin (Strongylocentrotus droebachiensis; ISF Trading, ing solution. Of course, the underlying basis of motile force Marine Biology Laboratory, Woods Hole, MA, USA) sperm were obtained and rotational motion is the basic adenosine triphosphate by intracoelomic injection of 0.5 M KCl. Eluted sperm were suspended in artificial seawater (ASW) containing 486 mM NaCl, 30 mM MgSO ,10mM (ATP)/dynein motor; calcium modulates this motor and other 4 MgCl2, 10 mM KCl, 10 mM CaCl2, 2.5 mM NaHCO3, and 10 mM HEPES components of the flagellar axoneme in ways that are not yet (pH 8.0). understood. In summary, we find that coitus induces oviductal flow In Vitro Sperm Rheotaxis that clears the oviduct of cellular debris and provides rheo- Sperm rheotactic movement was analyzed in convective and capillary flow. 5 tactic guidance for sperm. CatSper-mediated calcium entry For convective flow, 2 ml of culture medium and 1–2 3 10 mouse sperm or 3 5  associated with capacitation and subsequent hyperactivated 2–4 10 human sperm were added to the 37 C chamber (Delta T culture dish controller; Bioptechs). M2 or HEPES-HTF medium (92 mM NaCl, motility potentiates and regulates the sperm rotation that is 2 mM CaCl2, 4.7 mM KCl, 0.2 mM MgCl2, 0.37 mM KH2PO4, 25 mM NaHCO3, crucial to guidance in the graded, complex fluid flow in the 18.3 mM Na lactate, 2.78 mM glucose, 0.33 mM Na pyruvate, 0.4% [w/v] oviduct. bovine serum albumin [BSA], and 10 mM HEPES [pH 7.4]) was used for mouse or human sperm, respectively. In some experiments, the medium Experimental Procedures was supplemented with 0.3% (w/v) methylcellulose (MC) (M0512, 4,000 cP in 2% solution; Sigma). Sperm swimming 3–5 mm from the rim were re- Mating and Collection of Uterine Fluid corded by CCD camera (4 or 10 3 objective, mouse or human sperm, All animal experiments were performed according to procedures approved respectively; KP-F31SCL; Hitachi) after a 10 min preincubation period that by the Institutional Animal Care and Use Committee of Boston Children’s allowed spontaneous dissociation of sperm clumps and stabilized convec- Hospital. Mice were housed in a 12 hr light (7 a.m.–7 p.m.)/dark cycle. Follic- tive flow. For control (no flow) and capillary flow experiments, 1 ml of ular growth and ovulation were stimulated in CD-1 female mice (Charles medium was overlaid by 1 ml of mineral oil and covered by a heated glass River) at 8 to 9 weeks of age by intraperitoneal (i.p.) injection of pregnant lid (Bioptechs) to inhibit convection. A glass pipette of 1.2 mm (OD) rectan- mare serum gonadotropin (5 IU, PMSG; Calbiochem) at 6:30 p.m., followed gular glass capillary (w50 mm [h] 3 w1 mm [w]) was placed on the bottom 48 hr later by i.p. injection of human chorionic gonadotropin (5 IU, hCG; of the chamber, and the media was gravity fed at 1.5 ml/hr. Mouse sperm 5 Calbiochem). In some experiments, BrCr (10 mg/kg, B2134; Sigma) was (1–2 3 10 ) were transferred to the heating chamber and analyzed for  subcutaneously injected 11 hr before mating. The mating period for sperm rheotaxis at a constant 37 C. For sea urchin sperm, 2 ml of ASW 6 B6D2F1 males was set between 6:20 and 7:00 a.m. the morning after hCG and 1–2 3 10 sperm were added to a 35 mm culture dish precoated with injection; the end of the mating period was t = 0. In some experiments, 0.1% gelatin to prevent sperm surface adhesion. ASW containing 1 mm  atosiban (4 mg/kg) or tolvaptan (4 mg/kg) was injected i.p. just after mating. microbeads (Micromer; Micromod) was perfused at 3 ml/hr (22 C). The uterine horn was tied and dissected out with the oviduct and ovary, and uterine fluid was collected by gently squeezing the cut end of the uterus with Measurement of Sperm Rheotaxis forceps. The uterine fluid was the weight difference before and after fluid Sperm motion in 3 s intervals was recorded by CCD at 60 frames/s, collection. VR2 receptor antagonists for oxytocin (atosiban) or vasopressin processed using digital image processing software (XCAP; EPIX), and (tolvaptan) yielded 143 (659, SEM, n = 4) and 85 (616, SEM, n = 6) mg, analyzed using ImageJ. Swimming trajectories were via Computer Assisted respectively, of uterine fluid 8 hr after mating. Sperm Analysis (CASA; http://rsbweb.nih.gov/ij/plugins/casa.html). Sperm Sperm Rheotaxis 451

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