Will sperm cells exhibit chemotaxis towards sucrose?

Will sperm cells exhibit chemotaxis towards sucrose?

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I was wondering if sperm cells will exhibit chemotaxis towards sucrose? One of my friends told me that sperm cells die in presence of salts. So I guess sperms will move away from salt. Can someone give me any reference related to this area.

UPDATE (Answer for why sucrose?):

So I should have made things more clear. I was thinking to do an experiment by taking some sperm cells and its effects on sugar and salt and then to see it under a foldscope microscope. Since I am not a biologist, I wanted some guidance. The common available substances which came to my mind was sugar and salt.

If you google "sperm chemotaxis sucrose" you learn some things of interest. Notably, wikipedia's article on sperm chemotaxis indicates that the important chemoattractants for sperm (the things that attract sperm via chemotaxis) are various signaling molecules given off the by the egg.

It is not clear why you would necessarily think that sucrose would attract sperm or that salt would repel them. In the first case (sucrose), it is not obvious why sperm would expect to see sucrose. If I remember correctly, sucrose is actually pretty rare in animal physiology except as an energy source in plants that animals eat; and sucrose is largely broken down into monosaccharides before it even leaves the small intestine, so it's not like it's circulating in the body. For instance, a lab protocol for studying animal sperm chemotaxis suggests adding sucrose as a densifying agent that doesn't interfere with chemotaxis, suggesting that sperm are pretty independent of sucrose. In the second case (salt), it is not obvious that sperm would necessarily avoid the things that kill them. Those things may be correlated with doing their job! Most sperm cells are expected to die in the human reproductive strategy.

In plain terms: there are huge numbers of sperm which are competing really hard with each other to find and fertilize an egg, not to find sucrose or to avoid salt. The chemotaxis that sperm exhibit will tend to help them accomplish this goal. So it seems (according to wikipedia) that they follow the signals that say 'egg', not the ones that say 'sugar'.

Update: Sorry, only just saw update on Q. Thanks for the clarification- sounds like fun! I've been curious about the foldscopes.

If what you want is to do an experiment, then I think you've actually got a great start with knowing first that salt interferes with their motility. That is a control experiment, e.g. the very first thing you should do, to make sure that the world works the way other people say it should. (First rule of experimental science: never trust anyone!). Look up a protocol which suggests a sperm-inhibitory concentration of salt (ideally kosher salt, not table salt which has a bunch of other stuff like iodine in it). Put sperm in that concentration of salt and then also in a medium where they should show chemotaxis (the protocol I linked to could be helpful here). See if you can reproduce the salt inhibition, ideally in a medium that is otherwise identical to the medium that allows chemotaxis.

Next, try other stuff. Unfortunately, chemoattractants are probably proteins/small molecules you can't buy at the supermarket. But you could try to get creative. I can't ethically recommend that you try to find a human source, because that gets weird pretty quick, though it is the obvious suggestion (I assume you are talking about human sperm). Potentially there is an animal source for such things which isn't too expensive.

However, you could say to hell with chemoattractants and chemotaxis, and try to just understand sperm motility. You could try other osmotic shocks, with simple cheap chemicals like glycine. It looks like there is some literature that calcium ions affect sperm motility. Or you could play with pH, using vinegar or lye, or temperature. I bet that all of those affect sperm motility (probably for the worse). Or you could try the effects of various weird hippie herbs from the health food store. It sounds like you are just looking for an easy, fun experiment, and if that's your goal then I suggest you try a bunch of things and see what happens. That way you're more likely to find something interesting.

If what you want is a more elaborate experiment, then I think it is worth your time to sit down and try to read up on how sperm work, and then design an experiment that really clearly tests a hypothesis. I am not an expert here, but the Yoshida article I link to looks like it has a good literature review.

Hope that helps.

Even When Faint, Ovary Scent Draws Sperm Cells

Mice are known to have a keen sense of smell, but it's not just their noses that are adept at picking up a scent, a new study shows.

In this week's Analytical Chemistry, scientists at Indiana University Bloomington report biochemical machinery that allows mouse sperm cells to follow the weakest of scents. Even when ovary extracts were diluted 100,000 times, some sperm cells still found their mark.

"Sperm are known to exhibit chemotaxis toward extracts from various female reproductive organs, but the role of chemotaxis in reproduction is not known," said IUB Associate Professor of Chemistry Stephen C. Jacobson. "The chemicals that actually attract sperm have not been identified. Systematic study of various compounds released by the female reproductive organs under various conditions might further our understanding of these processes."

Understanding why, how and when sperm are attracted to ovaries may help scientists understand problems with human conception.

"Defects in sperm chemotaxis may be a cause of infertility, and consequently, sperm chemotaxis could potentially be used as a diagnostic tool to determine sperm quality or as a therapeutic procedure in male infertility," Jacobson said.

The project is a collaboration between research groups led by Jacobson and IUB Distinguished Professor of Chemistry Milos V. Novotny. Their work led to the development of a "flow-through" device, a sort of liquid treadmill for sperm cells, which allows researchers to follow the lateral movement of sperm as the cells swim through a liquid medium.

The device feeds three streams of liquid into a single chamber. Beyond the chamber, flows are split into three exit streams. Flow rate can be regulated simply by raising or lowering the height of the source medium, in this case a buffer solution. The researchers affixed a microscope and camera to the device and then recorded video of the sperm during assays.

"We combined in a microfluidic device the ability to generate a chemical gradient with transporting sperm cells to evaluate sperm chemotaxis," Jacobson said. "The use of microfluidic devices appears to be an ideal approach for precisely controlling the chemical gradient and accurately tracking chemotactic events. This combination led to greater repeatability in the experimental conditions over the course of the assays, which is currently not possible with conventional assays. These results are an important first step toward having an easy-to-use platform to rapidly evaluate and quantify chemotaxis."

The researchers hope their device will help other scientists more accurately examine the chemotaxis of all types of cells. The method also eliminates the phenomenon of "trapping," which causes study results to become ambiguous.

"The ability to differentiate chemotaxis from trapping helps to determine whether sperm were attracted to the test substance or the swim velocity was reduced close to the test substance," Jacobson said. "In the latter case, the test substance may have had a negative influence on the sperm, resulting in suppression of their movement."

Novotny is the Lilly Chemistry Alumni Chair in the Indiana University Bloomington Department of Chemistry, where he also directs the Institute for Pheromone Research.

IUB scientists Sachiko Koyama and Dragos Amarie led the experiments and were assisted by Helena A. Soini. The study was supported by Indiana University and the Lilly Chemistry Alumni Chair Fund. Analytical Chemistry is a journal of the American Chemical Society.

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Echinoderms, Part B

Hussein Hamzeh , . U. Benjamin Kaupp , in Methods in Cell Biology , 2019

1.1 The signaling pathway

Sperm chemotaxis has been primarily studied in sea urchins, but also in other marine invertebrates ( Carre & Sardet, 1981 Guerrero et al., 2010 Kaupp, 2012 Shiba, Baba, Inoue, & Yoshida, 2008 Ward, Brokaw, Garbers, & Vacquier, 1985 ). Sea urchins live in colonies on the sea floor and release eggs and sperm into the seawater for external fertilization. Upon spawning, the gametes are dispensed on a macroscopic scale by water flow. Sea urchin eggs are about 75–150 μm in diameter. To enlarge their effective target volume by several orders of magnitude, eggs release species-specific chemoattractant peptides (8–15 amino acids in length). The chemoattractant from the sea urchin A. punctulata, named resact, is composed of 14 amino-acid residues. An estimate suggests that sperm become attracted to the egg from a distance of about 0.5 cm ( Kashikar et al., 2012 ).

To provide a comprehensive account of all the molecules involved goes beyond the scope of this review. For a more detailed summary, we refer to previous reviews on A. punctulata ( Kaupp & Alvarez, 2016 Kaupp & Strünker, 2017 Wachten, Jikeli, & Kaupp, 2017 ) or Strongylocentrotus purpuratus sperm ( Darszon, Guerrero, Galindo, Nishigaki, & Wood, 2008 Darszon, Nishigaki, Beltran, & Trevino, 2011 Nishigaki et al., 2014 ). The most important signaling components are shown in Fig. 1 A . Binding of resact to a chemoreceptor located along the sperm flagellum activates a cellular signaling pathway. The chemoreceptor belongs to the family of membrane-spanning guanylate cyclases (GC), which synthesize the cellular messenger 3′,5′ cyclic guanosine monophosphate (cGMP) ( Garbers et al., 1988 Kaupp et al., 2003 ). The first signaling event is a rise of cGMP. In turn, cGMP opens K + -selective cyclic nucleotide-gated (CNGK) channels, and K + ions leave the cell ( Bönigk et al., 2009 Galindo, de la Vega-Beltrán, Labarca, Vacquier, & Darszon, 2007 Strünker et al., 2006 ). The ensuing hyperpolarization—the membrane potential (Vm) at rest is about − 50 mV—evokes a simultaneous increase of intracellular pH (pHi) and Na + concentration ([Na + ]i) via a sperm-specific, voltage-gated Na + /H + exchanger (sNHE) ( Lee & Garbers, 1986 Nomura & Vacquier, 2006 Seifert et al., 2015 Wang, King, Quill, Doolittle, & Garbers, 2003 Windler et al., 2018 ). The alkalization shifts the voltage dependence of the sperm-specific Ca 2 + channel CatSper (cation channel of sperm) to more negative voltages. This shift allows CatSper to open during recovery from hyperpolarization ( Seifert et al., 2015 ). The ensuing rise of the intracellular Ca 2 + concentration ([Ca 2 + ]i) modulates the flagellar beat. Recovery from hyperpolarization involves an inward Na + current carried by hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels ( Gauss, Seifert, & Kaupp, 1998 Seifert et al., 2015 ), closure of CNGK channels upon cGMP hydrolysis by a phosphodiesterase (PDE), or a combination of both. Restoration of Ca 2 + baseline levels is accomplished via a Na + /Ca 2 + /K + exchanger (NCKX) and a Ca 2 + -ATPase (PMCA) ( Gunaratne & Vacquier, 2006 Su & Vacquier, 2002, 2006 ). Fig. 1 B shows four principal signals during chemotaxis: a change in Vm, pHi, [Na + ]i, and [Ca 2 + ]i measured in the stopped-flow device as explained later in the text. The superimposed fluorescence responses illustrate the sequence of signaling events: First hyperpolarization, followed by simultaneous H + export and Na + import, and finally a rise of Ca 2 + .

Fig. 1 . Model of signaling pathway controlling chemotactic steering of A. punctulata sperm. (A) Binding of the chemoattractant resact to the chemoattractant receptor (GC) stimulates synthesis of cGMP, which opens K + channels (CNGK) and hyperpolarizes the cell. Hyperpolarization activates a sodium/proton exchanger (sNHE) the ensuing intracellular alkalization shifts the voltage dependence of the Ca 2 + channel CatSper to more negative membrane potentials. During recovery from hyperpolarization, probably facilitated by HCN1/HCN2 channel activity, CatSper channels open and Ca 2 + flows into the cell. Finally, Ca 2 + is extruded and basal levels are restored by a Na + /Ca 2 + -K + exchanger (NCKX) and a Ca 2 + pump (PMCA). (B) Signals represent changes in Ca 2 + (blue, Fluo-4), Vm (red, FluoVolt), pHi (green, pHrodo Red), and Na + (black, ANG2). Sperm were first mixed with ASW and then stimulated by photo-release of cGMP from DEACM-caged cGMP. Signals were recorded from sperm of the same animal and then aligned at the UV pulse.

Panel (A): Modified after Kaupp, U.B., &amp Strünker, T. (2017). Signaling in sperm: More different than similar. Trends in Cell Biology, 27, 101–109.

Mouse sperm exhibit chemotaxis to allurin, a truncated member of the cysteine-rich secretory protein family

Allurin, a 21 kDa protein isolated from egg jelly of the frog Xenopus laevis, has previously been demonstrated to attract frog sperm in two-chamber and microscopic assays. cDNA cloning and sequencing has shown that allurin is a truncated member of the Cysteine-Rich Secretory Protein (CRISP) family, whose members include mammalian sperm-binding proteins that have been postulated to play roles in spermatogenesis, sperm capacitation and sperm–egg binding in mammals. Here, we show that allurin is a chemoattractant for mouse sperm, as determined by a 2.5-fold stimulation of sperm passage across a porous membrane and by analysis of sperm trajectories within an allurin gradient as observed by time-lapse microscopy. Chemotaxis was accompanied by an overall change in trajectory from circular to linear thereby increasing sperm movement along the gradient axis. Allurin did not increase sperm velocity although it did produce a modest increase in flagellar beat frequency. Oregon Green 488-conjugated allurin was observed to bind to the sub-equatorial region of the mouse sperm head and to the midpiece of the flagellum. These findings demonstrate that sperm have retained the ability to bind and respond to truncated Crisp proteins over 300 million years of vertebrate evolution.


► Mouse sperm exhibit directed motility to allurin, an amphibian chemoattractant. ► Oregon green-conjugated allurin binds to mouse sperm in a region-specific pattern. ► Allurin stimulates a modest increase in beat frequency. ► Mouse sperm do not exhibit velocity increases in response to allurin. ► Sperm have retained the response to allurin over 300 million years of evolution.

Results and Discussion

To test the function of calaxin in regulation of sperm motility in chemotaxis, we used an inhibitor of neuronal calcium sensor family proteins, repaglinide, which specifically binds to calaxin in sperm flagella (Fig. S1) (13). We first asked whether calaxin plays a critical role in sperm chemotaxis. During chemotactic movements, sperm show a unique turning movement associated with a flagellar change to an asymmetric waveform, followed by a straight-ahead movement (11). We observed sperm chemotactic movement toward a glass capillary filled with SAAF in the absence and presence of repaglinide ( Fig. 1A ). Sperm in control artificial sea water (ASW) with 0.5% (vol/vol) solvent (DMSO) showed very strong chemotaxis toward the glass capillary. However, sperm in the ASW containing 150 µM repaglinide did not exhibit the unique turn movement and showed less-effective chemotaxis ( Fig. 1A ). Linear equation chemotaxis index (LECI) (11) analysis quantitatively showed significantly decreased chemotactic property promoted by repaglinide at 𾄀 µM ( Fig. 1B ). Sperm-swimming velocity showed no dramatic change following repaglinide treatment ( Fig. 1C ). One hypothesis is that loss of chemotactic behavior by repaglinide could be caused by an effect on KATP channels (13). However, treatment with glibenclamide, a specific KATP channel inhibitor, had no effect on chemotactic behavior ( Fig. 1B ). These data suggest that calaxin is essential for sperm chemotaxis.

Repaglinide inhibits sperm chemotaxis. (A) Sperm trajectories toward a SAAF-filled capillary (red) in the absence (Upper) and presence (Lower) of 150 µM repaglinide. (Scale bar, 100 µm.) Trajectories of three representative sperm are shown (Right). (B) Quantitation of chemotaxis using LECI. n = 12�. (C) Repaglinide does not significantly alter sperm swimming velocity during chemotaxis. n = 30. (D) Pseudocolor display of [Ca 2+ ]i as sperm swim toward a chemoattractant in the absence (DMSO) or presence of 150 µM repaglinide. Sperm trajectories and the position relative to chemoattractant (red) are shown in pseudocolor display of flagellar part (Right). (E) Maximum Fluo-8H fluorescent intensity of flagellar part during chemotaxis. Repaglinide treatment does not alter the maximum [Ca 2+ ]i. n = 12�. Arrows in A and D indicate direction of movement. *P < 0.01 vs. 0 µM **P < 0.001 vs. 0 µM.

Sperm chemotaxis is accompanied by an oscillatory [Ca 2+ ]i increase, followed by changes of flagellar asymmetry (Movie S1). To exclude the possibility that repaglinide alters chemotaxis by inhibiting oscillatory [Ca 2+ ]i increase, we compared [Ca 2+ ]i dynamics visualized by Fluo-8H during sperm chemotaxis in the absence and presence of repaglinide. Sperm treated with repaglinide showed transiently increased [Ca 2+ ]i near the chemoattractant similar to control sperm ( Fig. 1D Movie S2). The average maximum intensity of Fluo-8H fluorescence during chemotaxis was not affected by repaglinide ( Fig. 1E ). These results suggest that repaglinide inhibition of flagellar waveform is not due to an effect on [Ca 2+ ]i, but to direct action on calaxin.

To understand how chemotaxis is inhibited by repaglinide, flagellar waveform asymmetry was analyzed in detail using a high-speed camera. In the chemotactic turn, sperm show a transient change in flagellar asymmetry ( Fig. 2A Movie S3). Sperm treated with 150 µM repaglinide continued to exhibit transient flagellar asymmetry, but the trajectory of movement was unstable due to incomplete turning, resulting in less-effective chemotactic movement (Movie S4). Intriguingly, repaglinide-treated sperm could not sustain an asymmetric waveform and rapidly returned to a symmetric form, in contrast to control sperm, which showed strong asymmetry during one turn ( Fig. 2A , asterisks Movies S3 and S4). Detailed analysis revealed that repaglinide-treated sperm showed multiple asymmetric changes during one turn ( Fig. 2B , arrowheads Fig. 2C ), and the duration of the asymmetric state was decreased ( Fig. 2 B, Center, and D ). Such a decrease in duration could result in loss of a strong turn essential for chemotactic movement. The flagellar wave is composed of a large principal bend (P-bend) and a smaller reverse bend (R-bend) (9) (see Fig. S3). Asymmetry of flagellar waveform at the chemotactic turn was accompanied by both an increase of P-bend curvature and a decrease in R-bend curvature. Repaglinide-treated sperm achieved waveform asymmetry similar to that of control sperm ( Fig. 2B , Right), and as a result the maximum asymmetric indices were the same as those seen in control sperm ( Fig. 2E ). Decreased duration of the asymmetric waveform and the resulting restoration of symmetric waveform led to weak orientation of sperm movement to the chemoattractant.

Repaglinide treatment reduces sustainability of the asymmetric flagellar waveform at chemotactic turns. (A) Sequential images of sperm. Asterisks correspond to first one or two arrowheads in B. (B) (Left) Sperm trajectories in one turn movement. Arrows indicate direction of movement. Arrowheads indicate points of asymmetry. (Center) Asymmetric index. Raw data in dots are smoothed (solid line). (Right) Maximum flagellar curvatures of principal (blue) and reverse (red) bend during one chemotactic turn. (C) The number of asymmetries during one chemotactic turn is increased by repaglinide. n = 8�. (D) Duration of one asymmetry is significantly reduced by repaglinide. n = 12�. (E) Maximum asymmetric index during chemotaxis is not significantly altered by repaglinide. n = 6�. *P < 0.05 vs. 0 µM, **P < 0.01 vs. 0 µM.

Next, to examine the direct effect of repaglinide on flagellar axonemes, we used a model in which sperm were demembranated with 0.04% Triton X-100 and reactivated by 1 mM ATP. Various concentrations of Ca 2+ were added to the reactivating solution, and sperm waveform was analyzed. Reactivated sperm showed Ca 2+ -dependent changes in asymmetry of the flagellar waveform, as previously reported (8). Sperm flagella showed a more asymmetric waveform in solutions containing high Ca 2+ concentration ( Fig. 3 A and B ). Analysis of asymmetric indices at various [Ca 2+ ]i indicated that flagellar asymmetry became significant at greater than 10 𢄦 M Ca 2+ ( Fig. 3B ). However, flagellar bending of reactivated sperm treated with 150 µM repaglinide was attenuated at high Ca 2+ concentrations, although flagellar bending propagated at low concentrations of Ca 2+ ( Fig. 3C ). Comparison of bending curvature along the flagellar length at Ca 2+ concentrations between 10 � M (pCa10) and 10 𢄥 M (pCa5) showed that repaglinide treatment greatly impaired bend propagation, especially at the distal portion of flagella at pCa5 ( Fig. 3C ). A similar effect was observed by specific antibody against calaxin ( Fig. 3D ). This wave attenuation occurred in both the P-bend and R-bend at pCa5 and in the P-bend at pCa10 ( Fig. 3 C and D ). Glibenclamide treatment had no effect on flagellar amplitude at both low and high Ca 2+ concentrations (Fig. S2). A calmodulin inhibitor W-7 slightly lowered the P-bend curvature, but it caused no significant attenuation of flagellar curvature (Fig. S2), indicating that attenuation of flagellar curvature is due to specific inhibition of calaxin, not that of CaM-like proteins in the axoneme.

Calaxin regulates propagation of asymmetric flagellar bending by suppressing dynein-driven microtubule sliding. (A) Flagellar bending pattern at pCa10 (Left) and pCa5 (Right). Twenty bending forms were chosen every 5 ms. Flagellar curvature is plotted against the distance from the base of flagellum (Lower). Data from 20 waveforms are overwritten. (B) Flagellar asymmetric indices of flagella of a demembranated sperm model were plotted at various Ca 2+ concentrations. n = 18�. *P < 0.05 vs. pCa10, **P < 0.001 vs. pCa10. (C and D) Flagellar bending pattern and the curvature at pCa10 (Left) and pCa5 (Right) in the presence of 150 µM repaglinide (C) or anti-calaxin antibody (D). (Bottom) Maximum flagellar curvature against distance from the base of flagellum is plotted for P-bends and R-bends. n = 30�. *P < 0.01, **P < 0.001.

Calaxin has three Ca 2+ -binding motifs (EF-hand) (amino acids 62�, 98�, and 151�) (12). The association of calcium with calaxin was investigated using isothermal titration calorimetry (ITC Fig. 4A ). The ITC data for Ca 2+ could be fitted to a three-sites sequential binding model, consistent with the motif prediction ( Fig. 4A ). Two of the three EF-hand motifs exhibited endothermic binding (∆H1 = 8.0 kcal/mol and Ka1 = 2.3 × 10 5 M 𢄡 ∆H2 = 5.1 kcal/mol and Ka2 = 2.2 × 10 5 M 𢄡 ), and one was an exothermic site (∆H3 = 𢄣.4 kcal/mol and Ka3 = 3.5 × 10 4 M 𢄡 ). Positive enthalpy reflects hydrophobic interactions, as in the cases of the Ca 2+ binding to calmodulin (14) and (+)-abscisic acid (ABA) binding to PYL1 (15).

Ca 2+ binding to calaxin suppresses dynein-driven microtubule translocation. (A) Isothermal titration calorimetry showing three binding sites for Ca 2+ in calaxin. (Bottom) Location of three EF-hand Ca 2+ -binding motifs in calaxin. (B) Sequential dark-field images of microtubule translocation at pCa10 or pCa5 in the presence or absence of calaxin. Arrows indicate the direction of translocation. Arrowheads represent the minus (yellow) or plus (green) ends of microtubules. (C) Velocity of microtubule translocation. (Upper) Calaxin drastically suppresses translocation at pCa5. n = 73�. (Lower) Repaglinide (n = 40�), anti-calaxin antibody (n = 107�), and a mutation in EF-hand 2 of calaxin (n = 89�) cancel the calaxin-mediated suppression of microtubule translocation. Open bar, control closed bar, presence of repaglinide, anti-calaxin antibody, or a calaxin mutant. *P < 0.01, **P < 0.001. (D) Proposed model of calaxin function in sperm chemotaxis. A chemoattractant induces Ca 2+ influx and increased [Ca 2+ ]i triggers asymmetry of the flagellar waveform. Ca 2+ binding results in a conformational change in calaxin, its association with the dynein motor domain, suppression of microtubule sliding, and propagation of an asymmetric wave necessary for turn movement.

Finally, because calaxin is a strong candidate for a direct regulator of dynein motor activity (12), we asked if calaxin modulates sliding of polymerized singlet microtubules by purified outer arm dynein in vitro. After outer arm dynein was attached to a glass slide in a chamber, microtubules were added in the presence of 1 mM ATP, and microtubule translocation was recorded ( Fig. 4B Movies S5, S6, S7, and S8). Outer-arm dynein from Ciona sperm translocated microtubules at 4.6 ± 1.5 μm/s at pCa10. Increasing the concentration of Ca 2+ to pCa5 had a small effect on translocation velocity ( Fig. 4C ). However, addition of calaxin significantly reduced the velocity of microtubule translocation at pCa5 ( Fig. 4C ). Repaglinide cancelled the suppression of microtubule translocation at pCa5 ( Fig. 4C ). A specific antibody against calaxin also cancelled the suppression effect of calaxin ( Fig. 4C ). To disrupt Ca 2+ binding of calaxin, we prepared a calaxin mutant with E118A substitution in EF-hand motif 2 ( Fig. 4A ). The E118A mutant of calaxin showed no suppression of microtubule translocation even at pCa5 ( Fig. 4C ).

Analysis of Chlamydomonas mutants indicates that outer-arm dyneins are essential for conversion of waveform asymmetry in response to changes in Ca 2+ concentration (16 �). Calaxin binds to the β heavy chain of outer-arm dynein (12, 19) and suppresses microtubule sliding at high Ca 2+ concentrations ( Fig. 4 B and C ). Such suppression is likely required to propagate the asymmetric bend. In fact, both P- and R-bends of demembranated sperm are attenuated by treatment with the calaxin inhibitor repaglinide ( Fig. 3C ) or by anti-calaxin antibody ( Fig. 3D ). During chemotaxis of repaglinide-treated sperm, the asymmetric waveform does not propagate but becomes prematurely symmetric ( Fig. 2A ). It is generally accepted that flagellar bend propagation results from alternate sliding of doublet microtubules driven by dyneins. Mechanical feedback from one bend affects microtubule sliding in an adjacent bend (20). Therefore, suppression of microtubule sliding by calaxin could affect the adjacent bend to propagate an asymmetric waveform ( Fig. 4D ).

Conversion of asymmetrical waveform in Chlamydomonas flagella occurs at � 𢄦 M Ca 2+ in wild-type but not in outer-armless mutants (17). ATP-sensitive microtubule binding of Chlamydomonas outer-arm dynein is critical between � 𢄦 M and � 𢄥 M Ca 2+ (21). Although basal level of [Ca 2+ ]i is too low (㰐 𢄧 M) to estimate by using Fluo-8H in Ciona sperm, maximum asymmetric index during chemotactic turn movement ( Fig. 2E ) was comparable to that of demembranated sperm at � 𢄥 M Ca 2+ ( Fig. 3B ), suggesting that the maximum [Ca 2+ ]i during chemotactic turn is � 𢄥 M. This suggestion is compatible with the fact that [Ca 2+ ]i increases from 10 𢄧 to 10 𢄦 M by the chemoattractant speract in sea urchins (22). Suppression of microtubule sliding in vitro is significant at � 𢄥 M ( Fig. 4C ) and is consistent with both Ca 2+ response in sperm flagella ( Fig. 3 ) and hydrophobic interaction formed by Ca 2+ binding to two EF-hand motifs, as suggested from ITC ( Fig. 4A ). These results further support the idea that Ca 2+ -dependent suppression of microtubule-translocation activity of dynein by calaxin is a prerequisite for turn movement during sperm chemotaxis ( Fig. 4D ). Thus, Ca 2+ -mediated regulation of outer-arm dynein is thought critical for proper propagation of asymmetric flagellar waveform. The present study demonstrates not only the essential role of calaxin in sperm chemotaxis, but also the significance of outer-arm dynein in Ca 2+ -dependent regulation of asymmetric flagellar bend. Because calaxin is commonly present in metazoan cilia and flagella (12), further studies are likely to lead to important discoveries in the Ca 2+ -dependent regulation of various cilia-mediated processes.

Will sperm cells exhibit chemotaxis towards sucrose? - Biology

Behavioral traits of sperm are adapted to the reproductive strategy that each species employs. In polyandrous species, spermatozoa often form motile clusters, which might be advantageous for competing with sperm from other males [1]. Despite this presumed advantage for reproductive success [2, 3], little is known about how sperm form such functional assemblies. Previously, we reported that males of the coastal squid Loligo bleekeri produce two morphologically different euspermatozoa that are linked to distinctly different mating behaviors [4]. Consort and sneaker males use two distinct insemination sites, one inside and one outside the female’s body, respectively. Here, we show that sperm release a self-attracting molecule that causes only sneaker sperm to swarm. We identified CO2 as the sperm chemoattractant and membrane-bound flagellar carbonic anhydrase as its sensor. Downstream signaling results from the generation of extracellular H + , intracellular acidosis, and recovery from acidosis. These signaling events elicit Ca 2+ -dependent turning behavior, resulting in chemotactic swarming. These results illuminate the bifurcating evolution of sperm underlying the distinct fertilization strategies of this species.

Graphical Abstract


► Small, sneaky squid males produce spermatozoa that swarm to CO2 ► Sneaker, but not consort, sperm acidify their intracellular pH due to extracellular acidification by carbonic anhydrase ► Recovery from intracellular acidosis evokes Ca 2+ -dependent chemotactic swarming ► Swarming by sneaker sperm might be an evolutionary adaptation to mating tactics

Benefits of Microscale

Using a microfluidic device for these experiments is beneficial because it helps capture a more realistic environment in which to study the chemotaxis of the sperm. The sizes of fallopian tubes can range from 7 to 12 cm. in length, but the diameter of tube is normally less than 1 cm. Therefore, it makes sense that this kind of assay would be performed on a device that is relatively of the same scale. While the channel sizes for our device are definitely much smaller than that of the correct anatomical structure, the use of a microscale device will facilitate more accurate results than say an assay that is much larger than that of anatomical structures. Furthermore, it is unlikely that larger models would be able to efficiently capture what we are trying to assess. The gradients in larger devices would take longer to diffuse, and it would potentially take longer for sperm to detect. In a microfluidic assay, the gradients will form rather quickly, and it should take no time at all for the sperm to detect the gradients and to migrate more quickly.

Results and Discussion

Attractant Plumes Surrounding Eggs.

Field measurements within giant kelp forests (Macrocystis pyrifera) previously characterized the mixing properties of fluid into which abalone naturally spawn (13). These determinations specified the range of fluid-dynamic conditions for testing in present laboratory trials. Shears were 4.8 to 13.4 s −1 in adjacent open habitats, in contrast to 0.3 to 2.4 s −1 within the native crevices and under ledges where adult red abalone live and spawn (13). Spawning abalone thus aggregated at sites where water motion was substantially retarded (Fig. 1).

Theoretically, surface areas and volumes of egg-derived tryptophan plumes peaked in still water or in weak shears and decreased thereafter (Fig. 2 and Table S1). Weak shears (0.1–0.5 s −1 ) and slow flows [Pe of 1.5–7.5 Peclet number is a dimensionless ratio, reflecting flow speed (i.e., advection) relative to coefficient of molecular diffusion for l -tryptophan) resulted in elevated concentrations that decreased with increasing distance from an egg. The plumes thus expanded along the principal flow axis, relative to diffusion alone (i.e., still water Fig. 2B and Table S1). In contrast, strong shears (2.0–10.0 s −1 ) and fast flows (Pe of 30–151) rapidly reduced tryptophan concentrations below threshold in all but a very small region near the eggs (Fig. 2 E and G). Consequently, plume-maximizing shears were those most closely simulating flows in native spawning habitats (13).

Theoretical concentration distributions (nmol L −1 , in pseudocolor) of tryptophan surrounding red abalone eggs (black spheres) in still-water and in Taylor–Couette flows. Each plot is a 2D slice, cut through the center of an egg (AG). White arrows denote flow velocity vectors (speeds and directions). The x axis is parallel to the direction of flow and the y axis is orthogonal to flow, but parallel to the direction of shear. Tryptophan plumes were produced through 3D numerical simulations that used a coupled convection-diffusion model, taking into account egg rotation rate at each shear, and assuming a constant and continuous tryptophan release over the entire egg surface for 4 min at 0.18 fmol egg −1 min −1 (SI Materials and Methods). Model parameters (flow speed and direction, fluid shear, attractant release rate and diffusion coefficient, egg diameter and rotational velocity, water temperature) accurately portrayed conditions in our current experiments on sperm behavior, gamete encounter rates, and fertilization success. (Scale bar, 200 μm.)

Effects of Chemical Communication and Fluid Shear on Sperm Behavior.

Results of Taylor–Couette flow experiments strongly supported the theoretical predictions, validating the physical model of tryptophan plume dynamics (Materials and Methods provides details on Taylor–Couette apparatus and experimental protocols). Sperm swam faster and navigated directly toward egg surfaces within the predicted plumes (Fig. 3 A and B and 4 A and C). In contrast, male gametes positioned outside of the plumes swam slower and did not orient significantly with respect to an egg (Figs. 3 A and C and 4 F and H). Shear exerted a strong modulatory effect on sperm behavior (Tables S2 and S3). Swim speed and orientation toward an egg peaked at the weakest shears tested (0.1–0.5 s −1 ) and then decreased as shear strengthened. At 2.0 to 10.0 s −1 , flow-generated torques increasingly overwhelmed sperm behavior (13). These higher shears prevented male gametes from swimming actively across streamlines. Sperm aligned parallel to streamlines and cells tumbled at frequencies predicted by theory for passively transported particles (13). Whereas weak shears promoted, strong shears inhibited sperm locomotory performance and suppressed the attractant plume of egg-derived chemical signals.

(A) Representative swimming paths of individual red abalone sperm surrounding conspecific eggs in FSW as a function of shear. For each experimental treatment, a dashed line denotes the predicted behavioral threshold concentration (3 × 10 −10 mol L −1 ). This line demarcates the theoretical active space (i.e., closer to egg) from inactive space (i.e., further from egg) of an attractant plume. Active space was defined as the portion of a plume maintaining tryptophan greater than the threshold level that caused faster sperm swimming and orientation with respect to a chemical gradient. Small circles are successive digital images of sperm heads captured at 0.033-s intervals, and each arrowhead denotes the direction of travel for an individual cell. Sperm displacement caused by flow was subtracted on a frame-by-frame basis, so each computer-generated path reflects the actual track swum. To eliminate selection bias, a random numbers generator was used in choosing representative paths for each flow treatment. Orientation rosettes show distributions of directional tracks by sperm populations positioned inside (B) or outside (C) the active spaces of hypothesized tryptophan plumes. For B and C, complete data sets, not representative paths, were used to establish distributions and in calculating mean unit vector lengths (r) and angular headings (θ) relative to a line between each sperm head and the center of an egg. Sperm moving directly toward an egg would follow a 0° heading. A Rayleigh test (z-value) was used to compare each mean direction against a uniform circular distribution, and to calculate the P value. Sperm orientation toward an egg was significant inside the predicted active space at 0, 0.1, 0.5, 1.0, and 2.0 s −1 (V test: u ≥ 2.65, n ≥ 26 P < 0.04, all comparisons).

Effects of fluid shear on direction of sperm swimming with respect to an egg (A and F) (as described in more detail in Fig. 3), direction of sperm swimming with respect to flow (B and G), sperm translational swim speed (C and H), sperm encounter rate with a theoretical, tryptophan active space surrounding an egg (D), and sperm–egg encounter rate (E). Male gametes were imaged while swimming either “inside” or “outside” the active spaces of theoretical tryptophan plumes. Complete data sets, not representative paths, were used to establish mean unit vector lengths (r) with respect to an origin in A, B, F, and G. Experiments were performed in the presence of FSW alone (solid line) or with addition of active or denatured tryptophanase (dotted or dashed lines, respectively). Each dependent variable was described as a function of log-shear, using least-squares regression to establish the best fit (F tests for FSW and denatured enzyme treatments: F ≥ 7.39, df ≥ 1, 116 P < 0.001, all comparisons). Symbols are mean values (±SEM), and error bars are smaller than symbol sizes in some cases.

Effects of Chemical Communication and Fluid Shear on Sperm–Egg Encounter Rate and Fertilization Success.

Straighter and faster paths need not indicate that chemically mediated behavior increases encounter rates, or ultimately enhances fertilization success. As a function of shear, magnitudes of sperm swim speed and orientation (relative to an egg surface), male–female gamete contact rates, and percentages of fertilized eggs, all were highly correlated (Pearson product–moment correlation, r 2 ≥ 0.73, df = 6 P < 0.05, all comparisons Figs. 4 and 5 and Tables S2–S5). Thus, fertilization success could be forecasted accurately from sperm swimming behavior alone.

Effects of fluid shear on fertilization success (i.e., percentage of fertilized eggs). Egg density was held constant (10 3 cells mL −1 ) and, in separate treatments, sperm density was tested at (A) 10 6 , (B) 10 5 , or (C) 10 4 cells mL −1 . Experiments were performed in the presence of FSW alone (solid line) or with addition of active or denatured tryptophanase (dotted or dashed lines, respectively). Fertilization success was described as a function of log-shear, using least-squares regression to establish the best fit (F tests: F ≥ 50.5, df ≥ 1, 58 P < 0.001, all comparisons). Symbols are mean values (±SEM), and error bars are smaller than symbols in some cases.

Similar trends emerged across all sperm treatments. The percentages of fertilized eggs peaked at 0.1 to 0.5 s −1 , and then decreased as shear increased. At sperm concentrations of 10 5 and 10 4 cells mL −1 , maximal percentages of fertilized eggs were approximately 1.5 times those measured in still water (Fig. 5 B and C). In contrast, the maximal value decreased (1.2 times) slightly at a higher sperm density (10 6 cells mL −1 ), as overall fertilization levels approached saturation (an asymptote of 100% eggs fertilized Fig. 5A). Compared with still water, fertilization success was elevated significantly at 0.1 to 0.5 s −1 , was approximately the same at 1.0 to 2.0 s −1 , and was depressed significantly at 4.0 to 10.0 s −1 (Fig. 5 and Table S5). Weak shears therefore promoted sperm chemoattraction as well as reproductive success.

As always, correlation does not imply causation. Whereas results showed a strong association between egg-derived attractant plumes and sperm behavioral performance, these experiments were not designed to show a cause-and-effect relationship. Consequently, we determined whether eliminating the chemical signal around eggs would prevent fertilization. Freshly spawned eggs and sperm were placed in Taylor–Couette chambers containing filtered seawater (FSW) as before, but now with addition of activated or denatured (i.e., boiled) tryptophanase (2 μg mL −1 ). This enzyme, when active, selectively digests free tryptophan in solution.

The addition of activated tryptophanase had profound consequences for sperm–egg interactions. First, the enzyme did not affect sperm membranes, receptors, or behaviors, or the proclivity of male or female gametes for fertilization (22). It did, however, extinguish the signal surrounding an egg, as evidenced by sperm inability to navigate within hypothesized plumes, even from a distance of 100 μm, or less (Fig. 6 and Tables S6 and S7). HPLC indicated no measurable accumulation of tryptophan in seawater, when both enzyme and eggs were present. Second, elimination of the tryptophan and sperm chemoattraction precipitated a significant decrease in gamete encounter rate and fertilization success (Figs. 4 and 5 and Tables S8 and S9). Conversely, there was no decay in sperm navigation (toward an egg) and swim speed, encounter rate, or fertilization with the denatured tryptophanase (Figs. 4 and 5, Fig. S2, and Tables S6–S9). Tryptophan release by eggs, therefore, was a causative agent and critical determinant of fertilization success.

Effects of fluid shear and tryptophanase on representative swimming paths of individual red abalone sperm (A) and on orientation distributions of directional tracks by sperm populations positioned inside (B) or outside (C) of theoretical tryptophan plumes surrounding eggs. All experimental procedures and analyses, except for enzyme addition, were the same as described for Fig. 3. A Rayleigh test (z-value) was used to compare each mean direction against a uniform circular distribution, and to calculate the P value. Sperm orientation toward an egg was not significant in still water and at each tested shear (V tests: u ≤ 1.38 P ≥ 0.39).

Sperm chemoattraction and fluid shear each had significant effects on fertilization dynamics. Which process plays the ascendant role? To answer this question, we performed a series of stepwise multiple regressions on the fertilization data. Taken as a whole, our experiments measured percentages of fertilized eggs over a wide range of shears (0–10 s −1 , from abalone spawning to open kelp forest habitats), sperm densities (10 4 –10 6 cells mL −1 , from sperm-limiting to sperm-saturating conditions), and in the presence of active or denatured tryptophanase. Shear—not chemoattraction—explained most (55–64%) of the variation in fertilization success at low sperm densities (10 4 and 10 5 sperm mL −1 Table S10). In contrast, at a high sperm density (10 6 mL −1 ), chemoattraction had a significantly greater impact (67% of variation in fertilization). Thus, shear dominated chemical communication only under limiting sperm conditions. Shear did not damage either sperm or eggs (13). Instead, acting to facilitate or inhibit, it modulated the strength of chemically mediated, gamete interactions.

Chemical Communication, Fluid Shear, and Evolution of Sexual Reproduction.

The evolution of gamete size and morphology is a major unsolved problem in reproductive biology. Under conditions in which reproductive success is chronically limited by sperm availability, adults and gametes are under selection for mechanisms that increase sperm–egg contact (25). One such mechanism could involve changes in the physical size of the egg, because enlarging the “target” increases the probability of sperm–egg collision (25 ⇓ –27). Models of evolution have focused, traditionally, on postzygotic consequences of egg size for larval or juvenile survivorship (28, 29). Another implication of the target size hypothesis, however, is that prezygotic benefits to fertilization could drive the evolution of egg size and, in turn, anisogamy (25).

To date, theoretical models of gamete size evolution have not considered effects of fluid motion on sperm–egg encounter probabilities. Because shear is a natural feature of nearly all reproductive habitats, it may exert strong selective pressure on gamete morphology. In fact, shear initiates egg rotation at an angular velocity directly proportional to gamete size (i.e., radius) (11, 13, 30). As a consequence of this rotation, fluid accelerates when it approaches an egg, compressing or closing streamlines and locally increasing shear stress near an egg surface. The likelihood of sperm “slipping” around an egg surface, rather than encountering it, increases significantly with rotation rate (13). Thus, a larger egg is not always a better target.

For red abalone, chemoattraction provides a cheap evolutionary alternative for increasing egg target size without enlarging cytoplasmic and/or cell volume. Egg cytoplasm is an expensive commodity. It contains a vast array of organic molecules and provides a rich biochemical environment for synthesizing natural products after fusion, as required in embryo development (31). In contrast, the free amino acid l -tryptophan is taken up by maternal abalone from a dietary source and incorporated directly in egg cytoplasm during oogenesis. Thus, signal production, as well as release (via diffusion), consumes little or no metabolic energy and expends less than 1% of total cytoplasmic tryptophan reserves (24). Whereas tryptophan acts as a sperm attractant, it also is a precursor for synthesizing many neurotransmitters and neuromodulators. As a metabolic substrate essential to the development of the larval nervous system, tryptophan could be an honest indicator of egg fitness for prospective sperm suitors (32). Furthermore, red abalone eggs stop releasing tryptophan as they age and become infertile (24). Our results, therefore, suggest that endogenous signaling pathways have been coopted for external communication, as an adaptation to increase the likelihood of reproductive success. Because egg signaling and sperm response are possibly tuned to meet specific fluid-dynamic constraints, shear may act as a critical selective pressure that drives gamete evolution and determines fitness.

Cell migration

Cell migration is a central process in the development and maintenance of multicellular organisms. Processes such as tissue formation during embryonic development, wound healing, and immune responses, all require the orchestrated movement of cells in particular directions to specific locations. Errors during this process have serious consequences, including intellectual disability, vascular disease, tumor formation and metastasis. An understanding of the mechanism by which cells migrate may lead to the development of novel therapeutic strategies for controlling, for example, invasive tumor cells.

Cells often migrate in response to specific external signals, including chemical signals and mechanical signals. Due to a highly viscous environment, cells need to permanently produce forces in order to move. Cells achieve active movement by very different mechanisms. Many less complex prokaryotic organisms (and sperm cells) use flagella or cilia to propel themselves. Eukaryotic cell migration typically is far more complex and can consist of combinations of different migration mechanisms. It generally involves drastic changes in cell shape which are driven by the cytoskeleton, for instance a series of contractions and expansions due to cytoplasmic displacement. Two very distinct migration scenarios are crawling motion (most commonly studied) and blebbing motility.

The migration of cultured cells attached to a surface is commonly studied using microscopy. As cell movement is very slow (only a few µm/minute), time-lapse microscopy videos are recorded of the migrating cells to speed up the movement. Such videos reveal that the leading cell front is very active with a characteristic behavior of successive contractions and expansions. It is generally accepted that the leading front is the main motor that pulls the cell forward.

Cell Migration: Phase images of BSC 1 cells migrating in a scratch assay in the absence of serum over a period of 15 hours.

The stochastic dance of circling sperm cells: sperm chemotaxis in the plane

Biological systems such as single cells must function in the presence of fluctuations. It has been shown in a two-dimensional experimental setup that sea urchin sperm cells move toward a source of chemoattractant along planar trochoidal swimming paths, i.e. drifting circles. In these experiments, a pronounced variability of the swimming paths is observed. We present a theoretical description of sperm chemotaxis in two dimensions which takes fluctuations into account. We derive a coarse-grained theory of stochastic sperm swimming paths in a concentration field of chemoattractant. Fluctuations enter as multiplicative noise in the equations for the sperm swimming path. We discuss the stochastic properties of sperm swimming and predict a concentration-dependence of the effective diffusion constant of sperm swimming which could be tested in experiments.

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GENERAL SCIENTIFIC SUMMARY Introduction and background. Sensing the environment and moving actively in it are fundamental aspects of life. In many species, sperm cells can sense chemical cues from the egg and as a response actively steer towards the egg. This phenomenon of sperm chemotaxis is commonly studied in a two-dimensional experimental setup. In the absence of chemoattractant, sperm cells swim along circular paths. In the presence of a chemoattractant concentration gradient, however, their swimming circles drift on average gradient-upwards. A pronounced variability of the swimming paths is usually observed.

Main results. We develop a theoretical description of sperm chemotaxis taking into account nonequilibrium fluctuations. We employ a stochastic differential geometric description of the noisy swimming paths. We make a prediction about the effective diffusion coefficient of sperm swimming circles which can be tested in experiments: in our theory, this diffusion coefficient depends on the chemoattractant concentration measuring this dependence should reveal properties of the chemotactic signaling network such as its sensitivity threshold.

Wider implications. We have studied a particular biological example of cell locomotion guided by cellular signaling sytems which have to cope with imperfect sensory inputs and signal processing and still navigate the cell in a robust way. Similiar demands apply not only to sperm cells, but to many active cellular processes guided by external signals and in particular the locomotion of many microorganisms.

Figure. Simulated sperm swimming path (green) in a concentration gradient of a chemoattractant (blue). The sperm cell is equipped with a cellular signaling system which elicits a noisy chemotactic swimming response. The resulting sperm swimming path can be described as a swimming circle whose center (red) moves stochastically with net drift gradient-upwards.

This article was amended on 19 December 2008 to include the email address for F Jülicher.

Watch the video: Chemotaxis (September 2022).


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