View metadata, citation and similar papers at core.ac.uk brought to you by CORE 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 Rheotaxis 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- taxis, and it was concluded to be a laboratory artifact [9]. In Background: In sea urchins, spermatozoan motility 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 chemotaxis, 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) Bromocriptine 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.