Moreover, we identify P2 receptor-dependent purinergic signaling pathways as physiological triggers of tubular contractions both in vitro and in vivo. When challenged with extracellular ATP, transport of luminal content inside the seminiferous tubules displays stage-dependent directionality. We thus suggest that paracrine purinergic signaling coordinates peristaltic recurrent contractions of the mouse seminiferous tubules to propel immotile spermatozoa to the rete testis.Spermatogenesis, the complex process of male germ cell proliferation, differentiation, and maturation, is the basis of male fertility. In the seminiferous tubules of the testes, spermatozoa are constantly generated from spermatogonial stem cells through a stereotyped sequence of mitotic and meiotic divisions. The basic physiological principles, however, that control both maturation and luminal transport of the still immotile spermatozoa within the seminiferous tubules remain poorly, if at all, defined. Here, we show that coordinated contractions of smooth muscle-like testicular peritubular cells provide the propulsive force for luminal sperm transport toward the rete testis. Using a mouse model for in vivo imaging, we describe and quantify spontaneous tubular contractions and show a causal relationship between peritubular Ca As sperm develop in the testis, the immature cells must make their way through a maze of small tubes known as seminiferous tubules. However, at this stage, the cells do not yet move the long tails that normally allow them to ‘swim’; it is therefore unclear how they are able to move through the tubules.Now, Fleck, Kenzler et al. have showed that, in mice, muscle-like cells within the walls of seminiferous tubules can create waves of contractions that push sperm along. Further experiments were then conducted on cells grown in the laboratory. This revealed that a signaling molecule called ATP orchestrates the moving process by activating a cascade of molecular events that result in contractions. Fleck, Kenzler et al. then harnessed an advanced microscopy technique to demonstrate that this mechanism occurs in living mice. Together, these results provide a better understanding of how sperm mature, which could potentially be relevant for both male infertility and birth control. Spermatogenesis ranks among the most complex, yet least understood developmental processes in postnatal life. Inside the seminiferous tubules, which represent the functional units of the testis, this intricate course of mass cell proliferation and transformation events generates haploid spermatozoa from diploid spermatogonial stem cells. The seminiferous epithelium is composed of Sertoli cells, each intimately associated with ≥30 germ cells at different developmental stages . SertoliAccumulating evidence implicates purinergic signaling in testicular paracrine communication. While the general picture is still incomplete, cell- and stage-specific testicular expression of different purinoceptor isoforms has been reported in Leydig cells , Sertoli2+ permeability and show distinct pharmacological profiles, ligand affinities, and desensitization kinetics  or couple to Gαi/o  (Members of the P2 purinoceptor family are activated by extracellular ATP . P2 recekinetics . G-prote and 14) . Notably and 14) . So far, and 14) .In mice, stimulation-dependent ATP secretion from both Sertoli and germ cells was reported  and may rete testis and epididymis for final maturation. Precisely regulated tubular transport mechanisms are, thus, imperative for reproduction.Along the seminiferous epithelium, spermatogenesis has been conceptualized by attribution of sequential cellular ‘stages’ , which pWhile bulk movement of luminal content has been anecdotally reported , no quanAccumulating data suggests that purinergic signaling constitutes a critical component of testicular paracrine communication , with Se2+ imaging in cultured TPCs revealed robust and repetitive cytosolic Ca2+ transients upon ATP exposure (2+ concentration ([Ca2+]e) to 100 nM, a concentration approximately equimolar to cytosolic levels, by adding an appropriate chelator/ion concentration ratio (1 mM EGTA/0.5 mM CaCl2). This treatment, which drastically diminishes the driving force for Ca2+ influx, did substantially reduce, but not abolish ATP response amplitudes , indicating a role for G protein-dependent Ca2+ release from internal storage organelles  as well as a genetically encoded Ca2+ indicator (GCaMP6f). The dual excitation ratiometric indicator fura-2 allows semi-quantitative Ca2+ measurements  promoter drives Cre-mediated recombination of loxP-flanked reporters (tdTomato (Ai14D mice) or GCaMP6f (Ai95D)) in smooth muscle cells and TPCs . Brief ATP exposure resulted in a peripheral band of Ca2+ activity at the edge of the tubule. Such signals usually coincided with a pronounced contractile motion of the seminiferous tubule  underlying ATP-dependent TPC contractions. First, we asked whether influx of external Ca2+ is involved in TPC force generation. Similar to in vitro observations  were still detected in the vast majority of cells/experiments  essentially abolished both ATP-dependent Ca2+ signals and contractions  influx of external Ca2+ via ionotropic P2X receptors is not sufficient to drive TPC signals and evoke contractions. Notably, our general finding of ATP-induced mouse TPC contractions is likely transferable to human peritubular cells. When primary human TPC cultures  extracellular ATP acts as a potent TPC stimulus that triggers seminiferous tubule contractions in situ, that (ii) P2X and P2Y receptors act in concert to mediate TPC responses to ATP exposure, that (iii), while P2X receptor-dependent external Cacultures  were expcultures . Moreovecultures .We hypothesized that ATP-induced tubular contractions could impact the transport of luminal fluid and spermatozoa. To test this, we custom-built a whole-mount macroscopic imaging platform, designed to allow both widefield and fluorescence time-lapse imaging of intact seminiferous tubules . In addi2+ signals in TPCs (2+ elevations appear to be limited to those areas directly exposed to ATP (ROI 0). We observed no such signals in adjacent tubule sections independent of the stimulated stage group or an ascending or descending stage direction  occur spontaneously in living animals, and must (ii) be triggered experimentally by ATP exposure in vivo. Thus, to investigate any in vivo relevance of our findings, we designed a custom-built 3D printed in vivo imaging stage  can be triggered experimentally by ATP exposure. Consequently, paracrine purinergic signaling in the mouse testis is a mediator of luminal sperm transport within the seminiferous tubule network.Finally, we examined if brief focal ATP stimulation also triggers peritubular Ca tubules , Video 8eriments . By conteriments . Taken tThe molecular and cellular mechanisms that control paracrine testicular communication have to a large extent remained controversial, if not elusive . For TPCtunica albuginea, using ATP as a (co)transmitter (The site(s)/cellular origin of testicular ATP release as well as the mechanism(s) that trigger ATP secretion in vivo currently remain elusive. The apparent absence of efferent nerve endings in the seminiferous tubules and interstitial tissue  suggestsnsmitter . Regulatnsmitter . Moreovensmitter , which wnsmitter . Thus, T2+ transients observed in response to ATP exposure in various testicular cell types ll types , the mosswelling  adding aNotably, extracellular ATP is rapidly degraded by ecto-nucleotidases , renderi2+ influx and P2Y (likely isoform 2) receptor-activated phospholipase Cβ-dependent Ca2+ release from the sarcoplasmic reticulum – the latter being critical and resembling the recently reported mechanism of vascular smooth muscle cell contraction in small pulmonary veins  vs. micromolar (P2X) range  receptor-dependent Cary veins  – providneration . P2X andX) range . It is pX) range . Regardieceptors . This wa2+ signals in vivo, focal ATP stimulation appears insufficient to trigger a regenerative Ca2+ wave in isolated tubules. We, thus, conclude that the observed directionality results from other, likely structural characteristics, for example anatomical features of stage group II and III tubules that favor a specific flow direction . We cannot, however, rule out that the use of large field-of-view/low numerical aperture objectives for ‘macroscopic’ imaging simply prevents the detection of low-amplitude Ca2+ signal spread.Whole-mount imaging of isolated seminiferous tubules reveals propagation of luminal content that extends beyond the confines of the stimulated/contracted area and displays stage-dependent directionality. While peristaltic contractions are driven by propagating wave-like Ca2+ signals in human TPCs in vitro  whether TPCs are coupled by gap junctions to display coordinated activity; (ii) whether and, if so, how the final ATP metabolite adenosine affects seminiferous tubule physiology; (iii) whether Rho/Rho kinase signaling pathways modulate TPC contractility as frequently observed in other smooth muscle cells ; (iv) wh current ; (vi) wh current ; (vii) w current ; (viii) cillator  providescillator , a mechacillator  and othecillator  have obs2 asphyxiation and decapitation using sharp surgical scissors. We used C57BL/6J mice  as well as offspring from crossing either SMMHC-CreERT2 (JAX #019079)  and recommendations by the Federation of European Laboratory Animal Science Associations (FELASA). When possible, mice were housed in littermate groups of both sexes (room temperature (RT); 12:12 hr light-dark cycle; food and water available ad libitum). If not stated otherwise, experiments used adult (>12 weeks) males. Mice were killed by CO#019079)  or 129S.#019079)  mice wit#019079)  or Ai14D#019079)  mice, reThe following solutions were used:1S) 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffered extracellular solution containing (in mM) 145 NaCl, 5 KCl, 1 CaCl2, 0.5 MgCl2, 10 HEPES; pH = 7.3 (adjusted with NaOH); osmolarity = 300 mOsm (adjusted with glucose).(2S) Oxygenated  extracellular solution containing (in mM) 120 NaCl, 25 NaHCO3, 5 KCl, 1 CaCl2, 0.5 MgCl2, 5 N,N-bis(2-hydroxyethyl)−2-aminoethanesulfonic acid (BES); pH = 7.3; 300 mOsm (glucose).(3S) Extracellular low Ca2+ solution containing (in mM) 145 NaCl, 5 KCl, 0.5 MgCl2, 10 HEPES; pH = 7.3 (NaOH); osmolarity = 300 mOsm (glucose); [Ca2+]free = ~110 nM  or ~12 nM .(4S) Oxygenated  extracellular solution containing (in mM) 120 NaCl, 25 NaHCO3, 5 KCl, 0.5 MgCl2, 5 BES; pH = 7.3; 300 mOsm (glucose); [Ca2+]free = ~110 nM  or ~12 nM .(5S) Gluconate-based extracellular solution containing (in mM) 122.4 Na gluconate, 22.6 NaCl, 5 KCl, 1 CaCl2, 0.5 MgCl2, 10 HEPES; pH = 7.3 (adjusted with NaOH); osmolarity = 300 mOsm (glucose).(6S) Standard pipette solution containing (in mM) 143 KCl, 2 KOH, 1 EGTA, 0.3 CaCl2, 10 HEPES ([Ca2+]free = ~110 nM); pH = 7.1 (adjusted with KOH); osmolarity = 290 mOsm (glucose).(7S) Gluconate-based pipette solution containing (in mM) 110 Cs gluconate, 30 CsCl, 2 CsOH, 1 EGTA, 0.3 CaCl2, 10 HEPES ([Ca2+]free = ~110 nM); pH = 7.1 (adjusted with CsOH); osmolarity = 290 mOsm (glucose).. If not stated otherwise, chemicals were purchased from Sigma . Cyclopiazonic-acid (CPA) and 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate (BzATP) triethylammonium salt was purchased from Tocris Bioscience . Fura-2/AM was purchased from Thermo Fisher Scientific . Final solvent concentrations were ≤0.1%. When high ATP concentrations (≥1 mM) were used, pH was readjusted.In some experiments Na-GTP (0.5 mM) was added to the pipette solution. Free CaFor focal stimulation, solutions and agents were applied from air pressure-driven reservoirs via an 8-in-1 multi-barrel ‘perfusion pencil’ . Changes in focal superfusion  were sof2+]e solutions (3S a4nd S) were applied via both the bath and perfusion pencil. To ensure depletion of Ca2+ stores by CPA we monitored intracellular Ca2+ levels during drug treatment (0.05 Hz frame rate). Transient CPA-dependent Ca2+ elevations lasted 10–40 min. After baseline Ca2+ levels were restored, cells/slices were again challenged with ATP. Control recordings, omitting CPA, were performed under the same conditions.Low . If not stated otherwise, holding potential (Vhold) was –60 mV.Whole-cell patch-clamp recordings were performed as described . Brieflysoftware  and corr1S) and imaged with an upright microscope  equipped for ratiometric live-cell imaging with a 150 W xenon arc lamp, a motorized fast-change filter wheel illumination system for multi-wavelength excitation, a CCD camera , and Leica LAS X imaging software. Ten to thirty cells in randomly selected fields of view were viewed at 20x magnification and illuminated sequentially at 340 nm and 380 nm (cycle time 2 s). The average pixel intensity at 510 nm emission within user-selected ROIs was digitized and calculated as the f340/f380 intensity ratio.Cultured mouse TPCs were imaged as described . Briefly2+ signals and tubular contractions, acute seminiferous tubule slices were bulk-loaded with fura-2/AM in the dark . After washing , slices were transferred to a recording chamber and imaged with an upright microscope . We installed a custom-built reflective shield beneath the recording chamber for parallel monitoring of fluorescence and reflected light. At 1 Hz imaging cycles, we thus recorded two 510 nm fluorescence images (340/380 nm excitation) and a ‘pseudo-brightfield’ reflected light image that allowed quasi simultaneous analysis of intracellular Ca2+ and tubular movement.For parallel recordings of intracellular Ca2+ levels during CPA incubation at low frequency to monitor Ca2+ release from the ER, but also prevent phototoxicity. Experiments were only conducted if (i) we detected a substantial gradual rise in intracellular Ca2+ upon CPA treatment, and if (ii) functional Ca2+ extrusion mechanisms ensured that Ca2+-dependent fluorescence signals returned to base level. The time-course of this Ca2+ release – Ca2+ extrusion process varied between samples and ranged between 5.3 and 44.0 min .To ensure effective store depletion by CPA treatment, we recorded intracellular Ca1S. Small membrane holes under the tubules and around a defined stimulation area allowed for (i) gentle fixation of the tubules and (ii) focal ATP perfusion of selected tubular regions by vacuum-generated negative pressure (80–180 mmHg) in the submembraneous chamber compartment and continuous suction of 1S from the top compartment. After visual determination of tubular stages (I – III)  or tubular contractions and sperm transport. For low-magnification brightfield or fluorescence imaging, we used a MacroFluo Z16 APO A system (Leica Microsystems) equipped with either a DFC450C camera and a PLANAPO 1.0x/WD 97 mm objective (brightfield) or with a monochrome DFC365FX camera and a 5.0x/0.50 LWD PLANAPO objective (fluorescence). Images were acquired at 1 Hz.Isolated tubules (>1 cm length) were placed onto a membrane within a custom-built 3D printed two-compartment recording chamber that was constantly superfused with I – III) , the per−1 body weight) to double-positive adult male offspring (SMMHC-CreERT2 x Ai95D) via daily intraperitoneal injections for five consecutive days. Mice were closely monitored for any adverse reactions to the treatment. Experiments were performed 2–5 weeks after the first injection. For surgery, mice were anesthetized with ketamine-xylazine-buprenorphine . First, we made an incision next to the linea alba in the hypogastric region, followed by a 5 mm incision into the peritoneum. One testis was gently lifted from the abdominal cavity. Its gubernaculum was cut and the testis – with the spermatic cord, its blood vessels and vas deferens still intact – was transferred to a temperature-controlled imaging chamber filled with extracellular solution , mounted on a custom-designed 3D printed in vivo stage ) or stimulus solution  and ATP (1 mM)), respectively, from beveled glass micropipettes onto the surface of seminiferous tubules.We administered tamoxifen . Tests and corresponding p-values that report statistical significance (≤0.05) are individually specified in the legends. Data were analyzed offline using FitMaster 2.9 (HEKA Elektronik), IGOR Pro 8 , Excel 2016 , and Leica LAS X  software. Dose-response curves were fitted by the Hill-equation. Time-lapse live-cell imaging data displaying both Ca2+ signals and tubular contractions were analyzed using custom-written code in MATLAB .All data were obtained from independent experiments performed on at least three days. Individual numbers of cells/tubules/experiments (n) are denoted in the respective figures and/or legends. If not stated otherwise, results are presented as means ± SEM. Statistical analyses were performed using paired or unpaired f340 + f380) and then applied the displacement vector field, computed by the registration algorithm, to both images (f340 and f380) separately. ROIs were defined manually at f340/f380 ratio of all pixels within a given ROI. When measuring Ca2+-dependent changes in GCaMP6f intensity, the fluorescence signal th time point as For quantitative image analysis, images from both reflected light and fluorescence time-lapse recordings were registered to their respective first image frame at time point flow strengthflow changeSeminiferous tubule contractions and transport of luminal content were visualized by reflected light microscopy of acute slices or whole-mount macroscopic tubule imaging, respectively. Data from both types of time-lapse recordings were analyzed and quantified as either flow strength or flow change (see below). For each frame at a given time point segments , we quanSCR_002285) (bleach correction plugin in histogram matching mode (GausBlur (five px radius) and Gaussian Blur3D ). We calculated flow change via the Gaussian Window MSE function . Tubule selection used the polyline tool (line width adjusted to tubule diameter). Selected tubules ranged from 200 µm to 3.4 mm length. Next, flow fields of individual tubules were straightened. Average movement intensity was calculated from transversal line profiles  and plotted as kymographs (space-time plots) to measure movement progression speed from linear regressions.For analysis of in vivo data, we employed a custom set of ImageJ macros utilizing build-in functions of Fiji-ImageJ (RRID:_002285) . Widefieing mode . Next, wGausBlur (five px radius); Gaussian Blur3D ), flow fields were calculated from the background signal. Again, flow change was calculated via the Gaussian Window MSE function . Time-lapse epifluorescence in vivo recordings were processed to isolate transient fluorescence signals from static background noise using custom ImageJ code with Fiji’s build-in functions .Multiphoton time-lapse imaging data was recorded in dual-channel mode, with (i) a target channel recording GCaMP6f fluorescence and some background signal (525\50 nm), and (ii) a background channel mainly recording autofluorescence (585\40 nm), allowing for background correction of the GCaMP6f signal using a dye separation routine. Slow constant movement in both channels was registered and removed to correct for steady drift. After Gaussian filtering ; Johannes Reisert (Reviewer #3).Thank you for submitting your article "ATP activation of peritubular cells drives testicular sperm transport" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife, it is essential to revise and extend both the Introduction and the Discussion and to improve data presentation according to the specific comments listed below. Furthermore, reviewer 3 would particularly like to see clarification of the roles of P2X vs. P2Y receptors and also the potential dichotomy of the spread of contraction vs. the spread of Ca signaling.All three reviewers agreed that this is an excellent paper that adds a lot of new information to the field. However, the reviewers also agreed that to make this paper suitable for the broad readership of Reviewer #1:The manuscript entitled "ATP activation of peritubular cells drives testicular sperm transport" by Davis Fleck and co-workers deals with the involvement of purinergic signaling in testicular sperm transport.To obtain their results the authors used several state-of-the-art methods. In many of the conducted experiments they skilfully combined some of these methods to obtain unprecedented results. The authors verified their results employing different testis preparations (from cultured cells to in vivo recordings) and using different calcium sensors. They developed a very elegant custom-built 3D printed imaging stage that allowed experiments in the testis of living mice.The whole study is very well conducted, the methodology is sound, the statistical analysis is well done, and the authors provide convincing evidence for all their claims. They unambiguously show that nucleotide-triggered coordinated contractions of smooth muscle-like testicular peritubular cells provide the propulsive force for luminal sperm cell transport along seminiferous tubules.The results are extremely interesting for the fields of reproductive biology and reproductive medicine, but also for the whole field of physiology. The obtained results will with no doubt significantly influence the future research in the relevant fields.I do feel, however, that prior to publication a few points (see below) need to be clarified. Clarifying this points will improve the comprehensibility and readability of the manuscript.2+ waves in the Abstract, but do not mention them again in the whole manuscript. This is a little disturbing. I expected to find more information about these waves throughout the manuscript. How do the authors define these waves? Please clarify.1) Abstract: The authors mention peritubular Ca2) Although I generally like concise introductions, I feel that in the present manuscript the Introduction is too short. The authors should give more information about the cellular composition of the seminiferous tubules, the stages of sperm cell maturation, and the current knowledge of sperm cell transport in the tubules. The authors should consider including a schematic figure that gives an easy to understand overview of the above points. This will make all the performed experiments and the whole story easier to understand, especially for readers that are not experts in the field of reproductive biology and medicine.3) Subsection “ATP triggers seminiferous tubule contractions” and corresponding Figure 4E-G: I have difficulties to understand what is shown here. It might be only me, but the authors should consider trying to explain this part of their results better.4) Subsection “ATP drives directional luminal transport”: The authors state that in their preparation it is possible to visually categorize the twelve seminiferous epithelium cycle stages. I have difficulties to identify them. The authors should consider giving more information what exactly this twelve cycle stages are and how they can be distinguished in the picture . Part of this information could be included in an overview figure (see my first point above).5) Subsection “ATP drives directional luminal transport”: In this subsection the authors state that their finding indicates that ATP acts as a local paracrine messenger that, by itself, is not sufficient to trigger a signal that propagates in a wave-like fashion along the tubules. They speculate that transport directionality in the tubules likely results from morphological characteristics rather than peristaltic contractility.What do the authors mean with "morphological characteristics" and how can these characteristics explain the transport directionality? Also, why is the transport directionality probably not connected to peristaltic contractility?6) Subsection “ATP induces tubular contractions in vivo”: In this subsection the authors state that the movement analysis along the length of digitally straightened tubules demonstrates wave-like unidirectional motions that propagate with high velocities. What factors do drive this propagation? The authors should consider adding some information.7) Discussion: I generally like concise discussions, but in my opinion in this manuscript also the Discussion, like the Introduction, is way too short. It is more like a short conclusion. Here a few points that could/ should be speculated about:– What could be the in vivo origin of ATP ?– What could be the trigger for the physiological ATP release?– Why do nucleotides act "only" as paracrine mediators and do not induce calcium waves like in other tissues?– The authors could speculate about how likely it is that the same mechanisms are present also in human testis.– The authors could speculate about how their results could help finding treatments for infertility and / or be useful for the development of male contraception.– etc.8) I had problems with Videos 5 and 8. Somehow they did not run on my computers. I know that.mov-files are often requested, but there are certainly better formats for online videos. It could be a problem that my computers have, but I recommend checking these videos. Videos in an MP4 format would probably be better.Reviewer #2:2+ and peritubular cells movement. In addition, conclusions throughout the manuscript are sound and figure legends are clear. Because this manuscript is relevant for different fields, the authors should improve the writing significantly. First, the Introduction is very superficial and does not elaborate on the rationale behind this manuscript. Moreover, the authors assume that reproductive biologists are familiar with purinergic signaling and avoid any explanation on the pathways. Although short introductions are usually welcome, this one in particular has only two lines more than the Abstract (21 vs. 19 lines). The Results section is better; however, there are many sections in which the authors give for granted knowledge on the system . Finally, if the Introduction was short, the Discussion is almost absent (only 15 lines). The Discussion is also superficial, repeating some sections from the Abstract and Introduction. As the authors said, the topic of smooth muscle regulation in the testis has been underexplored for many years, there is much to say on the relevance of their findings that cannot be written in such a short Discussion.This manuscript by Fleck et al. investigates the molecular mechanism regulating smooth muscle contractions in the testis. Overall, this is an excellent manuscript using innovative imaging techniques to visualize almost simultaneously intracellular Ca1) Abstract: "Here, we show that coordinated contractions of smooth muscle-like testicular peritubular cells provide the propulsive force for luminal sperm transport toward the rete testis and epididymis." This sentence should be toned down; I agree that this manuscript shows coordinated contractions of smooth muscle. This finding suggests a possible mechanism for the propulsive force required for sperm transport; however, the manuscript does not make a direct observation of the sperm movement through the rete testis and epididymis.2) In the Abstract, the authors claim "Consequently, our findings could have substantial pharmaceutical implications for both infertility treatment and / or male contraception." As the authors recognized at the end of the manuscript, this sentence is speculative and does not belong to the Abstract.3) Introduction: The Introduction is not informative. The authors should give information on the purinergic system and its role in regulating smooth muscle. They should also incorporate to the Introduction text that let the reader knows on the main signaling pathways involved in purinergic signaling as well as on the type of purinergic receptors, agonists and antagonists.4) Subsection “ATP is a potent TPC stimulus”, explain briefly the conclusion that P2X2 or P2X4 but not P2X7 are responsible for the currents observed.5) Subsection “ATP is a potent TPC stimulus”: Explain the rationale of the conclusion "this current likely results from P2Y receptor-mediated phosphoinositide turnover" Why? Also, the authors state that these cells are sensitive to UTP and dependent on the presence of intracellular GTP. How were these experiments designed? Which is the conclusion of these findings? In general, more rationale is needed to explain why the experiments were conducted. For example, in Figure 1, ivermectin and suramin are used without introducing the mode of action of these compounds.2+ was reduced to 100 nM to avoid the reader to go to Materials and methods while reading the Results.6) Subsection “ATP is a potent TPC stimulus”: Explain briefly how the Ca7) Subsection “ATP is a potent TPC stimulus”: The UTP action is mentioned; however, only later in the same paragraph, the authors state that UTP is a selective P2Y agonist.Overall, the results are clear; however, it is very difficult to follow the rationale from the Results section text. As written this section is not more helpful than the figure legend.8) Subsection “ATP is a potent TPC stimulus”. Give a rationale for the use of GCaMP6f, explain briefly the strategy.9) Subsection “ATP is a potent TPC stimulus”: Define Ai14D mice, define SMMHC.Subsection “ATP triggers seminiferous tubule contractions”: Briefly explain the high frequency rippling observed.10) Subsection “ATP triggers seminiferous tubule contractions”: Mention the pharmacological inhibitor used and the time used for the treatment to avoid the reader to go to the Materials and methods section. In Figure 4 legend, the authors mentioned that they use CPA (please define) and they state that they pre-incubate for >= 15 min., could the time frame be more precise?11) Subsection “ATP triggers seminiferous tubule contractions”: Conclusions 'a' to 'c' are clear. Regarding the last conclusion 'd', briefly re-state the evidence supporting it; without stating the evidence, this conclusion is not sound.12) Figure 4. This figure lacks explanation in the main text. The reader needs to carefully analyze Figure 4 legend to understand the logic of the experiments performed. A summary of each panel in the main text will be helpful and should be added. In particular, there is no explanation of panel H in the main text. This panel is difficult to follow even after careful reading of the figure legend. The authors should make a significant effort to explain their thought process to scientists that are not necessarily experts in the field of purinergic signaling.13) Discussion:The authors speculate that their findings on purinergic signaling in the testis can have implications for male infertility and/or contraception. This sentence is repeated from the Abstract. Although there is nothing wrong to mention some putative translational implication, as written this sentence is not connected with the rest of the manuscript. To leave this sentence in a final manuscript, the authors should elaborate a rationale on how these findings can be used for either improving male fertility or for contraception. On the other hand, this speculation is not an important part of the manuscript and does not belong to the Abstract.14) Overall, the Discussion section is short and superficial. There is almost no analyses of the findings; some of the questions that should be addressed are: How results in this manuscript relates to other works in the area? Which are the unknowns that will need further investigation (gap in the knowledge)? How the purinergic system in the testis can be related to clinical applications ? Which are the advances obtained in terms of imaging methodologies? How the authors think that the system is regulated: is it paracrine as they propose? If this is the case, what would be the signaling for the release of ATP? It is ok not to know the answer, it is not ok to avoid discussing these topics.15) In summary, this is an excellent manuscript; however, it is written as a lab report for internal consumption. In general, the Results section needs a little more explanation on the rationale behind the experimental strategy and some clarification on the results. On the other hand, the authors should make a significant effort to improve the Introduction and Discussion section.Reviewer #3:2+ signals in testicular peritubular cells (TPCs) and hence their contractions in a directional manner. The manuscript is mostly very clearly laid out, experiments are well designed using broad and also entirely novel and quite impressive methodologies for in vivo recordings. Their results are an exciting and important contribution to a field fraught with confusion and they will lead to a deeper and further understanding of transport in seminiferous tubules.The manuscript by Fleck and colleagues addresses the role of ATP and its receptors in sperm transport in the seminiferous tubules in the testis. They conclude that ATP, in a paracrine manner, drives intracellular Ca2+ signaling in TPCs is somewhat confusing. In Figure 1J, the authors demonstrate that the Ca2+ signal in TPCs originates both from extracellular sources (P2X mediated) as well as intracellular sources (P2Y mediated). But depletion of intracellular stores  seems to entirely eliminate the cellular Ca2+ signal, suggesting P2Y mediated signaling only? Also, given that P2Y-mediated Ca2+ signals do play a role, it might be worth considering moving data in Figure 1—figure supplement 1 into Figure 1 of the main paper to give them more prominence.1) The roles of P2X and P2Y receptors and their contribution to Ca2+ signals respectively following focal application of ATP, with contraction occurring in a stage-dependent directional manner, while the Ca2+ signal is confined locally to where ATP was applied with no spread. This seems puzzling as it raises the question of what signal drives the contractions  in the TPCs that contract (one assumes) without an apparent Ca2+ signal?2) Figures 5 and 6 describe directional luminal transport and TPC Ca2+ waves spreading from cell to cell ? While this might be outside the scope of the manuscript, it might be worth addressing this issue in the currently quite focused Discussion.3) Along similar lines, what signal drives the directionality of the contractions? ATP diffusing along the tubules? Or Ca Reviewer #1:[…] The results are extremely interesting for the fields of reproductive biology and reproductive medicine, but also for the whole field of physiology. The obtained results will with no doubt significantly influence the future research in the relevant fields.I do feel, however, that prior to publication a few points (see below) need to be clarified. Clarifying these points will improve the comprehensibility and readability of the manuscript.2+ waves in the Abstract, but do not mention them again in the whole manuscript. This is a little disturbing. I expected to find more information about these waves throughout the manuscript. How do the authors define these waves? Please clarify.1) Abstract: The authors mention peritubular Ca2+ waves” in the original manuscript (aside from the Abstract). This has now been corrected. We now show a representative Ca2+ wave that travels in a unidirectional manner along a tubule’s longitudinal axis . We use a sequence of successive single frames from time-lapse in vivo recordings (5 Hz image acquisition rate), in which we overlay a brightfield image with a pseudocolor fluorescence intensity image that indicates elevations in TPC Ca2+ concentration. The wave-like character also becomes apparent from an overlay of traces (fluorescence intensity over time) corresponding to adjacent regions-of-interest in the same tubule.Reviewer 1 makes a valid point. We failed to refer to the “CaAccordingly, we have now revised the Results and Discussion sections, which now read:2+ waves that travel at comparable speed and direction . Notably, the observed coordinated contractile movements…”Results: “…Movement analysis along the length of digitally straightened tubules demonstrates wave-like unidirectional motions that propagate with high velocities . These movements coincide with ‘macroscopic’ Ca2+ elevations in TPCs that propagate in a wave-like fashion, and (iii) can be triggered experimentally by ATP exposure.…”Results: “…Taken together, in vivo recordings demonstrate that robust recurrent seminiferous tubule contractions (i) occur spontaneously, (ii) are driven by cytosolic Ca2+ signals in vivo, focal ATP stimulation appears insufficient to trigger a regenerative Ca2+ wave in isolated tubules. We, thus, conclude…”Discussion: “…While peristaltic contractions are driven by propagating wave-like CaThe legend of Figure 7—figure supplement 2 has been revised accordingly.“Data and materials availability” subsection.The custom code used for image processing is made available by link in the 2) Although I generally like concise introductions, I feel that in the present manuscript the Introduction is too short. The authors should give more information about the cellular composition of the seminiferous tubules, the stages of sperm cell maturation, and the current knowledge of sperm cell transport in the tubules. The authors should consider including a schematic figure that gives an easy to understand overview of the above points. This will make all the performed experiments and the whole story easier to understand, especially for readers that are not experts in the field of reproductive biology and medicine.We thank reviewer 1 for raising this important point and for his insightful suggestions. We acknowledge his argument and, accordingly, we have now added “a schematic figure that gives an easy to understand overview of the above points”. This new schematic diagram is now introduced as Figure 1A and it illustrates both “cellular composition of the seminiferous tubules” and “the stages of sperm cell maturation.” The legend of Figure 1 has been revised accordingly.We have also added new text to the Introduction, which now reads: “The seminiferous epithelium is composed of Sertoli cells, each intimately associated with ≥30 germ cells at different developmental stages . […] After approximately 39 days (4.5 cycle repetitions), spermatogenesis completes with the release of immotile spermatozoa from the seminiferous epithelium into the lumen of the tubule (spermiation).”3) Subsection “ATP triggers seminiferous tubule contractions” and corresponding Figure 4E-G: I have difficulties to understand what is shown here. It might be only me, but the authors should consider trying to explain this part of their results better.Reviewer 1 emphasizes an important point. We apologize for the somewhat vague description of our approach and, accordingly, we have extensively revised the corresponding Results section.2+ is involved in TPC force generation. […] Together, these data strongly suggest that (i) extracellular ATP acts as a potent TPC stimulus that triggers seminiferous tubule contractions in situ, that (ii) P2X and P2Y receptors act in concert to mediate TPC responses to ATP exposure, that (iii), while P2X receptor-dependent external Ca2+ influx apparently boosts responses to ATP, P2Y receptor-mediated Ca2+ mobilization from the sarcoplasmic reticulum is necessary to evoke TPC responses, and consequently that (iv) influx of external Ca2+ via ionotropic P2X receptors is not sufficient to drive TPC signals and evoke contractions. Notably, our general finding…”We added several explanatory remarks to the relevant paragraphs, which now read: “First, we asked whether influx of external Ca2+, black; contractions, red) of responding TPCs / tubules, quantified as a function of stimulus, treatment, and Ca2+ sensor. Data (mean ± SEM) are normalized to the respective initial responses to ATP (10 µM) under control conditions ).”In addition, we added explanatory notes to the legend of Figure 4, which now reads: “(F) Signal amplitudes (Ca4) Subsection “ATP drives directional luminal transport”: The authors state that in their preparation it is possible to visually categorize the twelve seminiferous epithelium cycle stages. I have difficulties to identify them. The authors should consider giving more information what exactly this twelve cycle stages are and how they can be distinguished in the picture . Part of this information could be included in an overview figure (see my first point above).We thank reviewer 1 for pointing this out. Our wording has obviously been misleading. In fact, we cannot identify each of the twelve seminiferous epithelium cycle stages in the whole-mount preparation that we established. What we can do, however, is following the approach first proposed by Hess and De Franca, 2008, and categorize stages I-V as ‘early’, stages VI-VIII as ‘middle’, and stages IX-XII as ‘late’. The corresponding ‘stage groups I, II and III’ are characterized by different densities of luminal sperm that can be distinguished clearly by standard widefield microscopy.To clarify, we followed the expert’s advice and have now added this information to “an overview figure”  above). We now also refer to the three groups again in Figure 5A, using the same color code. Moreover, we have revised the Results that now reads:I).”“In addition, this setup enables visual categorization of the spermatogenic cycle into three distinct stage groups following published protocols  and allows precisely timed focal perfusion . […] When we analyzed ATP-induced movement in directly stimulated regions (each designated as region-of-interest (ROI) 0), comparing stage groups with a high (group II) versus a relatively low (groups I and III) amount of luminal sperm, we observed no difference in stimulation-dependent motion  Subsection “ATP drives directional luminal transport”: In this subsection the authors state that their finding indicates that ATP acts as a local paracrine messenger that, by itself, is not sufficient to trigger a signal that propagates in a wave-like fashion along the tubules. They speculate that transport directionality in the tubules likely results from morphological characteristics rather than peristaltic contractility.What do the authors mean with "morphological characteristics" and how can these characteristics explain the transport directionality? Also, why is the transport directionality probably not connected to peristaltic contractility?Reviewer 1 raises another important point that is also brought up by reviewer 3 (main points 2 and 3). It is thus becoming clear that we did not manage to communicate these results and, specifically, our interpretation in a clear and compelling way. We apologize and, accordingly, we have extensively revised both the corresponding Results text and the Discussion.𝑖𝑖) is different from the contraction measurements (flow strength 𝑠𝑠𝑖𝑖) conducted in Figures 3, 4 and 7. We describe the difference in the “Data analysis” subsection of the Materials and methods. We believe that this important distinction did not become sufficiently clear in the previous version of the manuscript. In a nutshell, the results shown in Figure 5 cannot be interpreted as evidence for propagating longitudinal contractions (nor do they argue against such a phenomenon). What the results do show is that (i) a local ATP stimulus provides the impulse to push luminal content – with or without triggering a peristaltic wave – and that (ii) for epithelial regions in stage group II, this movement displays directionality.In Figure 5, we show that focal application of ATP triggers contractions that translate into movement of luminal content. This read-out , we assume that, under the present experimental conditions, other features are involved. “Morphological characteristics” such as an anatomical setting in which Sertoli cells effectively function as ‘nonreturn valves’ could, for example, be involved (see below).In Figure 6, we then ask if the underlying mechanism might be a peristaltic contraction, which we do assume must be causally related to TPC Ca2+ wave in isolated seminiferous tubules could, of course, have several reasons, including any number of missing environmental factors present in the intact testis or simply the inability to register low-amplitude Ca2+ signals when using large field-of-view / low numerical aperture objectives. Importantly, we always detect coincident Ca2+ signals when monitoring seminiferous tubule contractions in vivo . Moreover, propagation of wave-like Ca2+ signals is also regularly observed in vivo .The absence of a regenerative Ca2+ signal – contraction coupling and propagation. Specifically, we have revised / added the following text:In the revised manuscript, we now make an effort to describe the data and their interpretation in clear terms in the Results. Moreover, we have added a dedicated paragraph to the Discussion that picks up on the issue of CaResults: “Third, we investigated if luminal movement is restricted to the area of stimulation or, by contrast, if fluid flow propagates beyond the directly stimulated tubule section. […] These findings demonstrate directionality of sperm transport upon focal purinergic TPC stimulation in isolated seminiferous tubules.”2+ signals in TPCs . […] However, local contractions generate sufficient force to move luminal content beyond the directly stimulated area and, in turn, directionality of flow along short-to-medium distances  is not critically dependent on peristaltic contractility.”Results: “As expected, ATP-induced tubule contractions also manifest as Ca2+ signal spread.”Discussion: “Whole-mount imaging of isolated seminiferous tubules reveals propagation of luminal content that extends beyond the confines of the stimulated / contracted area and displays stage-dependent directionality. […] We cannot, however, rule out that the use of large field-of-view / low numerical aperture objectives for ‘macroscopic’ imaging simply prevents the detection of low-amplitude Ca2+ oscillator  provides an endogenous pacemaker mechanism that acts independent of purinergic stimulation; and…”Discussion: “Among several remaining questions, future experimental efforts will have to address (i) whether TPCs are coupled by gap junctions to display coordinated activity; (ii) whether… […] …(viii) whether an additional cytosolic and / or membrane Ca6) Subsection “ATP induces tubular contractions in vivo”: In this subsection the authors state that the movement analysis along the length of digitally straightened tubules demonstrates wave-like unidirectional motions that propagate with high velocities. What factors do drive this propagation? The authors should consider adding some information.2+ elevations, which we now refer to several times in the manuscript and we also show a representative example . The detailed mechanisms underlying the spread of the Ca2+ signal and, therefore, the propagation of contractions / luminal content motion currently remain subject to debate and speculation. Accordingly, we have now added several relevant new paragraphs to the Discussion (see our response to point 7 below).Again, reviewer 1 makes a valid point that also refers to his points 1, 5, and 7 . As outlined in our response to these points, the primary factor that drives this propagation is wave-like TPC Ca7) Discussion: I generally like concise discussions, but in my opinion in this manuscript also the Discussion, like the Introduction, is way too short. It is more like a short conclusion.The reviewer makes an excellent suggestion. We acknowledge his argument and, therefore, we have now substantially revised the Discussion. Several new paragraphs have been added to address the reviewers concerns.Specifically, the following text has been added.Here a few points that could/ should be speculated about:– What could be the in vivo origin of ATP ?Discussion: “The site(s) / cellular origin of testicular ATP release as well as the mechanism(s) that trigger ATP secretion in vivo currently remain elusive. […] Thus, TPCs could themselves participate in regenerative nucleotide release.”– What could be the trigger for the physiological ATP release?Discussion: “During spermatogenesis, apoptosis is a vital process . […] In addition, ATP release has been observed in several cell types as a result of mechanical deformation, shear stress, stretch, or osmotic swelling  adding another putative mechanism of regenerative signaling in purinergic contraction control.”– Why do nucleotides act "only" as paracrine mediators and do not induce calcium waves like in other tissues?We have now addressed this point in the Discussion. For brevity, we refer to our response to concern 5 (see above).– The authors could speculate about how likely it is that the same mechanisms are present also in human testis.Discussion: “Translation of our findings from the mouse model to humans awaits further in-depth investigation. […] While a single layer of TPCs surrounds the mouse seminiferous tubules, the human tubular wall architecture is more complex, containing several TPC layers, substantial amounts of extracellular matrix proteins, and immune cells .”– The authors could speculate about how their results could help finding treatments for infertility and / or be useful for the development of male contraception.Discussion: “Impaired spermatogenesis in sub- / infertile men typically coincides with tubular wall remodeling and a partial loss of TPC contractility proteins has been reported in infertile men . […] Accordingly, pharmacological targeting of purinergic signaling pathways to (re)gain control of TPC contractility represents an attractive approach for male infertility treatment or contraceptive development.”8) I had problems with Videos 5 and 8. Somehow they did not run on my computers. I know that.mov-files are often requested, but there are certainly better formats for online videos. It could be a problem that my computers have, but I recommend checking these videos. Videos in an MP4 format would probably be better.We acknowledge the reviewer’s point. We have checked the.mov files on several PCs and found no difficulties. Yet, we have also rendered the video files as.mp4 and can provide them upon request should the editors prefer this format, which produces somewhat larger files.Reviewer #2:2+ and peritubular cells movement. In addition, conclusions throughout the manuscript are sound and figure legends are clear. Because this manuscript is relevant for different fields, the authors should improve the writing significantly. First, the Introduction is very superficial and does not elaborate on the rationale behind this manuscript. Moreover, the authors assume that reproductive biologists are familiar with purinergic signaling and avoid any explanation on the pathways. Although short introductions are usually welcome, this one in particular has only two lines more than the Abstract (21 vs. 19 lines). The Results section is better; however, there are many sections in which the authors give for granted knowledge on the system . Finally, if the Introduction was short, the Discussion is almost absent (only 15 lines). The Discussion is also superficial, repeating some sections from the Abstract and Introduction. As the authors said, the topic of smooth muscle regulation in the testis has been underexplored for many years, there is much to say on the relevance of their findings that cannot be written in such a short Discussion.This manuscript by Fleck et al. investigates the molecular mechanism regulating smooth muscle contractions in the testis. Overall, this is an excellent manuscript using innovative imaging techniques to visualize almost simultaneously intracellular Ca1) Abstract: "Here, we show that coordinated contractions of smooth muscle-like testicular peritubular cells provide the propulsive force for luminal sperm transport towards the rete testis and epididymis." This sentence should be toned down; I agree that this manuscript shows coordinated contractions of smooth muscle. This finding suggests a possible mechanism for the propulsive force required for sperm transport; however, the manuscript does not make a direct observation of the sperm movement through the rete testis and epididymis.towards the rete testis”, not “through the rete testis.” We believe that this is still a valid point to make. In conclusion, we acknowledge the reviewer’s argument and, accordingly, we have deleted the reference to the epididymis and rephrased the relevant sentence, which now reads:Reviewer 2 makes a valid point. We did not intend to claim that we “make a direct observation of the sperm movement through the rete testis and epididymis.” In fact, we are not able to make any statement that refers to the passage of sperm within the rete testis and epididymis. We would like to respectfully point out to the reviewer that we intentionally referred to “sperm transport “Here, we show that coordinated contractions of smooth muscle-like testicular peritubular cells provide the propulsive force for luminal sperm transport towards the rete testis.”2) In the Abstract, the authors claim "Consequently, our findings could have substantial pharmaceutical implications for both infertility treatment and / or male contraception." As the authors recognized at the end of the manuscript, this sentence is speculative and does not belong to the Abstract.We acknowledge the reviewer’s argument and, accordingly, we have now deleted this speculative sentence from the Abstract.3) Introduction: The Introduction is not informative. The authors should give information on the purinergic system and its role in regulating smooth muscle. They should also incorporate to the Introduction text that let the reader knows on the main signaling pathways involved in purinergic signaling as well as on the type of purinergic receptors, agonists and antagonists.Reviewer 2 emphasizes an important point. We agree that the Introduction, in its original form, may have been too superficial. Thus, we acknowledge the reviewer’s argument and, accordingly, we have now added “information on the purinergic system and its role in regulating smooth muscle” as well as on “the main signaling pathways involved in purinergic signaling”. The relevant paragraphs in the revised Introduction now read:“Accumulating evidence implicates purinergic signaling in testicular paracrine communication. […] Functionally, several studies have suggested purinergic paracrine control of gonadotropin effects on Leydig and Sertoli cells , including steroidogenesis and testosterone / 17β-estradiol secretion .”“Members of the P2 purinoceptor family are activated by extracellular ATP . […] So far, the most prominent role for a specific subunit in reproductive physiology has been attributed to P2X1, which is critical for vas deferens smooth muscle contraction and male fertility .”“In mice, stimulation-dependent ATP secretion from both Sertoli and germ cells was reported  and may itself be under endocrine control . […] Alternative ATP release pathways include connexin / pannexin hemichannels , transporters , voltage-gated  or large-conductance anion  channels, or even P2X7 receptors .”4) Subsection “ATP is a potent TPC stimulus”, explain briefly the conclusion that P2X2 or P2X4 but not P2X7 are responsible for the currents observed.We have added an explanatory section to the relevant Results paragraph, which now reads:“The specific biophysical and pharmacological profile of ATP-dependent transmembrane currents  strongly suggests functional expression of P2X2 and / or P2X4, but not P2X7 receptors. […] Ivermectin , an agent selectively potentiating P2X4 receptor currents , increased ATP-induced currents in a subpopulation of TPCs (n = 7/12), whereas suramin , a drug inhibiting P2X2, but not P2X4 receptors , inhibited a TPC subset (n = 10/18).”5) Subsection “ATP is a potent TPC stimulus”: Explain the rationale of the conclusion "this current likely results from P2Y receptor-mediated phosphoinositide turnover" Why? Also, the authors state that these cells are sensitive to UTP and dependent on the presence of intracellular GTP. How were these experiments designed? Which is the conclusion of these findings? In general, more rationale is needed to explain why the experiments were conducted. For example, in Figure 1, ivermectin and suramin are used without introducing the mode of action of these compounds.We thank reviewer 2 for pointing this out. We acknowledge his / her argument and, accordingly, we have extensively revised the corresponding Results paragraph, which now reads:2+ imaging in cultured TPCs revealed robust and repetitive cytosolic Ca2+ transients upon ATP exposure . […] Together, these data suggest that mouse TPCs functionally express both ionotropic and metabotropic purinoceptors.”“Notably, live-cell ratiometric CaThe modes of action of “ivermectin and suramin” have now also been described (please see our response to point 4 above).2+ was reduced to 100 nM to avoid the reader to go to Materials and methods while reading the Results.6) Subsection “ATP is a potent TPC stimulus”: Explain briefly how the Ca2+ concentration ([Ca2+]e) to 100 nM, a concentration approximately equimolar to cytosolic levels, by adding an appropriate chelator / ion concentration ratio (1 mM EGTA / 0.5 mM CaCl2). This treatment, which drastically diminishes the driving force for Ca2+ influx, did substantially reduce, but not abolish ATP response amplitudes .”This has been done. The relevant sentence now reads: “We next reduced the extracellular Ca7) Subsection “ATP is a potent TPC stimulus”: The UTP action is mentioned; however, only later in the same paragraph, the authors state that UTP is a selective P2Y agonist.This has now been corrected (please see our response to point 5 above).8) Subsection “ATP is a potent TPC stimulus”. Give a rationale for the use of GCaMP6f, explain briefly the strategy.This has now been done. The relevant sentence now reads:2+ reporters, a synthetic ratiometric Ca2+ sensor (fura-2) as well as a genetically encoded Ca2+ indicator (GCaMP6f). […] By contrast, conditional gene targeting via the Cre/Lox system“In parallel approaches, we employed two different fluorescent Ca allows TPC-specific expression of the single-wavelength indicator GCaMP6f.”9) Subsection “ATP is a potent TPC stimulus”: Define Ai14D mice, define SMMHC.This has been done. The relevant sentence now reads:T2 under control of the mouse smooth muscle myosin, heavy polypeptide 11 (a.k.a. SMMHC) promoter drives Cre-mediated recombination of loxP-flanked reporters (tdTomato (Ai14D mice) or GCaMP6f (Ai95D)) in smooth muscle cells and TPCs .”“Tamoxifen-induced transgenic expression of CreER10) Subsection “ATP triggers seminiferous tubule contractions”: Mention the pharmacological inhibitor used and the time used for the treatment to avoid the reader to go to the Materials and methods section. In Figure 4 legend, the authors mentioned that they use CPA (please define) and they state that they pre-incubate for >= 15 min., could the time frame be more precise?2+-ATPase, is now defined (subsection “ATP triggers seminiferous tubule contractions”).Cyclopiazonic-acid (CPA), a blocker of the sarco / endoplasmic reticulum Ca2+ imaging” section in Materials and methods, which reads:Regarding the duration of CPA treatment, we now added “more precise” information to the legend of Figure 4D . To explain the variability in incubation time, we now also added an explanatory paragraph to the “Fluorescence Ca2+ levels during CPA incubation at low frequency to monitor Ca2+ release from the ER, but also prevent phototoxicity. […] The time-course of this Ca2+ release – Ca2+ extrusion process varied between samples and ranged between 5.3 and 44.0 min .”“To ensure effective store depletion by CPA treatment, we recorded intracellular Ca11) Subsection “ATP triggers seminiferous tubule contractions”: Conclusions 'a' to 'c' are clear. Regarding the last conclusion 'd', briefly re-state the evidence supporting it; without stating the evidence, this conclusion is not sound.We thank reviewer 2 for raising this concern. We acknowledge his/her argument and, accordingly, we have revised the relevant Results paragraph that now reads:2+ influx apparently boosts responses to ATP, P2Y receptor-mediated Ca2+ mobilization from the sarcoplasmic reticulum is necessary to evoke TPC responses, and consequently – since store depletion essentially abolishes ATP-dependent signals – that (iv) influx of external Ca2+ via ionotropic P2X receptors is not sufficient to drive TPC signals and evoke contractions.”“Together, these data strongly suggest that (i) extracellular ATP acts as a potent TPC stimulus that triggers seminiferous tubule contractions in situ, that (ii) P2X and P2Y receptors act in concert to mediate TPC responses to ATP exposure, that (iii), while P2X receptor-dependent external Ca12) Figure 4. This figure lacks explanation in the main text. The reader needs to carefully analyze Figure 4 legend to understand the logic of the experiments performed. A summary of each panel in the main text will be helpful and should be added. In particular, there is no explanation of panel H in the main text. This panel is difficult to follow even after careful reading of the figure legend. The authors should make a significant effort to explain their thought process to scientists that are not necessarily experts in the field of purinergic signaling.Reviewer 2 makes a valid point. Accordingly, we have now made “a significant effort to explain” the rationale, particularly with respect to Figure 4H. The substantially revised Results paragraph that now reads:2+ signaling mechanism(s) underlying ATP-dependent TPC contractions. […] Notably, these UTP responses were statistically indistinguishable from the diminished ATP-dependent signals we observed under low [Ca2+]e conditions .”“We next investigated the Ca13) Discussion:The authors speculate that their findings on purinergic signaling in the testis can have implications for male infertility and/or contraception. This sentence is repeated from the Abstract. Although there is nothing wrong to mention some putative translational implication, as written this sentence is not connected with the rest of the manuscript. To leave this sentence in a final manuscript, the authors should elaborate a rationale on how these findings can be used for either improving male fertility or for contraception. On the other hand, this speculation is not an important part of the manuscript and does not belong to the Abstract.We agree and thank reviewer 2 for pointing this out. As mentioned above (see response to point 2) we deleted this sentence from the Abstract. In agreement with a request from reviewer 1 (point 7), we have now added “a rationale on how these findings can be used for either improving male fertility or for contraception” to the Discussion, which now reads:“Impaired spermatogenesis in sub- / infertile men typically coincides with tubular wall remodeling and a partial loss of TPC contractility proteins has been reported in infertile men . […] Accordingly, pharmacological targeting of purinergic signaling pathways to (re)gain control of TPC contractility represents an attractive approach for male infertility treatment or contraceptive development”.14) Overall, the Discussion section is short and superficial. There is almost no analyses of the findings; some of the questions that should be addressed are: How results in this manuscript relates to other works in the area? Which are the unknowns that will need further investigation (gap in the knowledge)? How the purinergic system in the testis can be related to clinical applications ? Which are the advances obtained in terms of imaging methodologies? How the authors think that the system is regulated: is it paracrine as they propose? If this is the case, what would be the signaling for the release of ATP? It is ok not to know the answer, it is not ok to avoid discussing these topics.We thank reviewer 2 for making this important point, which is in agreement with a concern raised by reviewer 1 (point 7). Therefore, we have now substantially revised the Discussion. Several new paragraphs have been added to address the reviewers concerns.Specifically, the following text has been added:How results in this manuscript relates to other works in the area? Which are the unknowns that will need further investigation (gap in the knowledge)?2+-gated Cl– current ; (vi) why periods of enhanced contractile activity are interrupted by longer quiescent episodes ; (vii) which additional or complementary roles in TPC physiology are played by previously proposed activators, including vasopressin, oxytocin, prostaglandins, and endothelin ; (viii) whether an additional cytosolic and / or membrane Ca2+ oscillator  provides an endogenous pacemaker mechanism that acts independent of purinergic stimulation; and (ix) whether, similar to vascular smooth muscle cells, some specific tone is maintained between contractions by spatial averaging of asynchronous oscillations , a mechanism that could explain the occurrence of spontaneous low-amplitude ‘vibratory’ movements and local indentations that we  and others  have observed.”Discussion: “Among several remaining questions, future experimental efforts will have to address (i) whether TPCs are coupled by gap junctions to display coordinated activity; (ii) whether and, if so, how the final ATP metabolite adenosine affects seminiferous tubule physiology; (iii) whether Rho/Rho kinase signaling pathways modulate TPC contractility as frequently observed in other smooth muscle cells ; (iv) what, if any, role is played by P2X receptor-dependent changes in membrane potential; (v) which function is served by the sustained CaHow the purinergic system in the testis can be related to clinical applications ?Discussion: “Translation of our findings from the mouse model to humans awaits further in-depth investigation. […] Accordingly, pharmacological targeting of purinergic signaling pathways to (re)gain control of TPC contractility represents an attractive approach for male infertility treatment or contraceptive development.”How the authors think that the system is regulated: is it paracrine as they propose? If this is the case, what would be the signaling for the release of ATP?Discussion: “The site(s) / cellular origin of testicular ATP release as well as the mechanism(s) that trigger ATP secretion in vivo currently remain elusive. […] This way, the combined action of P2X and P2Y receptors might equip TPCs with a broader ‘two-step’ stimulus integration range.”15) In summary, this is an excellent manuscript; however, it is written as a lab report for internal consumption. In general, the Results section needs a little more explanation on the rationale behind the experimental strategy and some clarification on the results. On the other hand, the authors should make a significant effort to improve the Introduction and Discussion section.We thank reviewer 2 for his / her thoughtful comments, which helped us create a better manuscript. As outlined above, we have now provided the required “clarification on the results” and we made “a significant effort to improve the Introduction and Discussion section.”Reviewer #3:2+ signaling in TPCs is somewhat confusing. In Figure 1J, the authors demonstrate that the Ca2+ signal in TPCs originates both from extracellular sources (P2X mediated) as well as intracellular sources (P2Y mediated). But depletion of intracellular stores  seems to entirely eliminate the cellular Ca2+ signal, suggesting P2Y mediated signaling only? Also, given that P2Y-mediated Ca2+ signals do play a role, it might be worth considering moving data in Figure 1—figure supplement 1 into Figure 1 of the main paper to give them more prominence.[…] (1) The roles of P2X and P2Y receptors and their contribution to Ca2+ release from the sarcoplasmic reticulum – appears puzzling. However, it is likely that P2X receptors act as signal boosters that mediate Ca2+-induced Ca2+ release, possibly via activation of ryanodine receptors . To clarify, we have added a new paragraph to the Discussion, which now reads:Reviewer 3 makes a valid point. At first glance, the role of P2X receptors – given the critical role of Ca2+-induced Ca2+ release, possibly via activation of ryanodine receptors . This way, the combined action of P2X and P2Y receptors might equip TPCs with a broader ‘two-step’ stimulus integration range.”“Excitation–contraction coupling in TPCs is poorly described. […] Therefore, it is likely that P2X receptors act as signal boosters that mediate Ca2+-activated Cl– current as new Figure 1—figure supplement 2.In addition, we acknowledge the reviewer’s argument that “it might be worth considering moving data in Figure 1—figure supplement 1 into Figure 1 of the main paper to give them more prominence.” Accordingly, we now show the relevant data  as new Figure 1M–P. For coherence, we now present data showing the Ca2+ signals respectively following focal application of ATP, with contraction occurring in a stage-dependent directional manner, while the Ca2+ signal is confined locally to where ATP was applied with no spread. This seems puzzling as it raises the question of what signal drives the contractions  in the TPCs that contract (one assumes) without an apparent Ca2+ signal?2) Figures 5 and 6 describe directional luminal transport and TPC CaReviewer 3 raises an important point that is also brought up by reviewer 1 . It is clear that we did not manage to communicate these results and, specifically, our interpretation in a clear and compelling way. We apologize and, accordingly, we have extensively revised both the corresponding Results text and the Discussion.𝑖𝑖) is different from the contraction measurements (flow strength 𝑠𝑠𝑖𝑖) conducted in Figures 3, 4 and 7. We describe the difference in the “Data analysis” subsection of the Materials and methods. We believe that this important distinction did not become sufficiently clear in the previous version of the manuscript. In a nutshell, the results shown in Figure 5 cannot be interpreted as evidence for propagating longitudinal contractions (nor do they argue against such a phenomenon). What the results do show is that (i) a local ATP stimulus provides the impulse to push luminal content – with or without triggering a peristaltic wave – and that (ii) for epithelia in stage group II, this movement displays directionality.In Figure 5, we show that focal application of ATP triggers contractions that translate into movement of luminal content. This read-out , we assume that, under the present experimental conditions, other features are involved. For example, Sertoli cell morphology could effectively cause them to function as ‘nonreturn valves’.In Figure 6, we then ask if the underlying mechanism might be a peristaltic contraction, which we do assume must be causally related to TPC Ca2+ wave in isolated seminiferous tubules could, of course, have several reasons, including any number of missing environmental factors present in the intact testis or simply our inability to register low-amplitude Ca2+ signals when using large field-of-view / low numerical aperture objectives. Importantly, we always detect coincident Ca2+ signals when monitoring seminiferous tubule contractions in vivo . Moreover, propagation of wave-like Ca2+ signals is also regularly observed in vivo .The absence of a regenerative Ca2+ signal contraction coupling and propagation. Specifically, we have revised / added the following text in the Results:In the revised manuscript, we now make an effort to describe the data and their interpretation in clear terms in the Results. Moreover, we have added dedicated paragraphs to the Discussion that pick up on the issue of Ca“Third, we investigated if luminal movement is restricted to the area of stimulation or, by contrast, if fluid flow propagates beyond the directly stimulated tubule section. […] These findings demonstrate directionality of sperm transport upon focal purinergic TPC stimulation in isolated seminiferous tubules.”2+ signals in TPCs . However, these Ca2+ elevations appear to be limited to those areas directly exposed to ATP (ROI 0). […] However, local contractions generate sufficient force to move luminal content beyond the directly stimulated area and, in turn, directionality of flow along short-to-medium distances  is not critically dependent on peristaltic contractility.”“As expected, ATP-induced tubule contractions also manifest as Ca2+ signal spread.”Discussion: “Whole-mount imaging of isolated seminiferous tubules reveals propagation of luminal content that extends beyond the confines of the stimulated / contracted area and displays stage-dependent directionality. […] We cannot, however, rule out that the use of large field-of-view / low numerical aperture objectives for ‘macroscopic’ imaging simply prevents the detection of low-amplitude Ca2+ oscillator  provides an endogenous pacemaker mechanism that acts independent of purinergic stimulation; and…”“Among several remaining questions, future experimental efforts will have to address (i) whether TPCs are coupled by gap junctions to display coordinated activity; (ii) whether… […] …(viii) whether an additional cytosolic and / or membrane Ca2+ waves spreading from cell to cell ? While this might be outside the scope of the manuscript, it might be worth addressing this issue in the currently quite focused Discussion.Along similar lines, what signal drives the directionality of the contractions? ATP diffusing along the tubules? Or CaReviewer 3 emphasizes another important point. As he stated, an in-depth analysis of the mechanism(s) that govern excitation–contraction coupling and its longitudinal spread in TPCs is “outside the scope of the manuscript” and warrants systematic full analysis and an independent publication. Nonetheless, we follow the reviewer’s advice to address “this issue in the currently quite focused Discussion.” Accordingly, we have revised the Discussion as outlined in our response to concern 2 (see above). In addition, we have now added several relevant paragraphs to the Discussion that now reads:“During spermatogenesis, apoptosis is a vital process . […] In addition, ATP release has been observed in several cell types as a result of mechanical deformation, shear stress, stretch, or osmotic swelling  adding another putative mechanism of regenerative signaling in purinergic contraction control.”“Notably, extracellular ATP is rapidly degraded by ecto-nucleotidases , rendering its interstitial half-life relatively short and, thus, narrowing its paracrine radius to a few hundred micrometers . Combined with its fast diffusion – approximately 1 μm in less than 10 ms  – extracellular ATP bears all characteristics of a fast paracrine agent in testicular communication .”“Excitation–contraction coupling in TPCs is poorly described. […] This way, the combined action of P2X and P2Y receptors might equip TPCs with a broader ‘two-step’ stimulus integration range.”


In this study, we aimed to examine the cellular mechanisms underlying ALI-induced [Ca2+]i increase in cultured urothelial cells. The ALI was created by stopping the influx of the perfusion but maintaining efflux. The [Ca2+]i increase was measured using the Ca2+ imaging method. The ALI evoked a reversible [Ca2+]i increase and ATP release in urothelial cells, which was almost abolished by GdCl3. The specific antagonist of the transient receptor potential vanilloid (TRPV4) channel (HC0674) and the antagonist of the pannexin 1 channel (10panx) both diminished the [Ca2+]i increase. The blocker of Ca2+-ATPase pumps on the endoplasmic reticulum (thapsigargin), the IP3 receptor antagonist (Xest-C), and the ryanodine receptor antagonist (ryanodine) all attenuated the [Ca2+]i increase. Degrading extracellular ATP with apyrase or blocking ATP receptors (P2X or P2Y) with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) significantly attenuated the [Ca2+]i increase. Our results suggest that both Ca2+ influx via TRPV4 or pannexin 1 and Ca2+ release from intracellular Ca2+ stores via IP3 or ryanodine receptors contribute to the mechanical responses of urothelial cells. The release of ATP further enhances the [Ca2+]i increase by activating P2X and P2Y receptors via autocrine or paracrine mechanisms.Urothelial cells have been implicated in bladder mechanosensory transduction, and thus, initiation of the micturition reflex. Cell deformation caused by tension forces at an air-liquid interface (ALI) can induce an increase in intracellular Ca The release of ATP from urothelial cells directly activates purinergic receptors on bladder afferent terminals to increase their excitability, thereby enhancing sensory function of the bladder  on urothelial cells is the first step in initiating mechanotransduction. These channels detect and transduce external mechanical forces into intracellular Cachannels ; transiechannels ; epithelchannels ; the Piechannels ; Piezo2 channels ; pannexichannels ; and conchannels .In vivo, changes in intravesical pressure or bladder wall tension are physiologically relevant mechanical stimuli for the urothelium. To simulate these stimuli in vitro, common strategies include stretching of urothelial cells cultured on coverslips using various customized devices .Several studies have reported that cells can be mechanically stimulated by creating an air-liquid interface (ALI). The ALI is produced by reducing the volume of liquid covering cells, and a thin film of liquid on the cells is drawn down to such proximity that tension forces at the ALI cause cell deformation . In the poration , to inve2+]i and ATP release from urothelial cells cultured on glass coverslips, and the responses were repeatable and did not affect cell viability. We aimed to investigate the underlying cellular mechanisms of mechanical stimulus-induced increase in [Ca2+]i with this easily-operated model. Our main focus was on the potential involving channels or receptors located either on the cell membrane or those associated with intracellular Ca2+ stores. We found that both Ca2+ influx via membrane-bound TRPV4 or pannexin 1 channels, and Ca2+ release from intracellular Ca2+ stores via IP3 and ryanodine receptors contribute to an ALI-induced [Ca2+]i increase. Furthermore, ATP released from urothelial cells also contribute to a [Ca2+]i increase via autocrine or paracrine mechanisms.In the present study, we developed another method to create an ALI in a perfusion cell chamber, by simply stopping influx, but maintaining efflux of the perfusion, until a thin layer of liquid was created on the urothelial cells. We found that the ALI can induce a remarkable increase in .g for 5 min. The cells were suspended in urothelial cell medium  with 1% PSF, centrifuged again, and resuspended in fresh media. Cells were plated on poly-L-lysine-coated glass coverslips and used for Ca2+ imaging, 48–96 h after dissociation.Urothelial cell cultures were performed as previously described . Rats we2PO4; 4 NaHCO3; 2 CaCl2; 1 MgCl2; 10 HEPES; and 5.6 glucose. The pH was adjusted with Tris base to 7.4, and osmolality was adjusted with sucrose to 320–325 mOsm. The influx of perfusion was driven by gravity, and the efflux was driven by a vacuum pump. Monolayer urothelial cells were immersed in HBSS when the influx and efflux flow rates were equally maintained, and no ALI was formed on the cells , the amount of HBSS solution in the chamber was reduced to a level, at which a thin liquid layer was formed covering the cells. Cells were mechanically deformed by the tension produced at the ALI, and fluorescence signals at 340 and 380 nm were altered inset of . The aboormation .2 and 5% CO2. Fura 2-AM was dissolved in HBSS. Ca2+ imaging was performed as described previously  for 30–40 min at 37°C in an atmosphere of 95% Oeviously . BrieflyThe ATP concentration was measured by using luciferin-luciferase bioluminescence. Samples (100 μl) of perfusate were collected 2 min before and immediately after ALI stimulation. A mixture of 100 μl luciferin-luciferase was added to a 100 μl sample, according to the manufacturer’s instructions using the Promega CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega). ATP detection was evaluated using the GloMax 20/20 Luminometer (Promega). Sample bioluminescence was compared to that of standard amounts of ATP used within the same concentration range. All samples were run in duplicate.Immunofluorescence staining was performed to identify the expression of TRPV4 and pannexin 1 in urothelial cells. The bladder tissue was frozen in standard cryomolds containing optimal cutting temperature (OCT) compound. The tissue was then sectioned, permeabilized with 0.5% Triton X-100 for 30 min, and blocked with 5% goat serum for 1 h. The primary antibodies used were rabbit polyclonal anti-TRPV4 antibody ; mouse monoclonal anti-cytokeratin AE1/AE3 antibody ; rabbit polyclonal anti-pannexin 1 antibody , and normal rabbit IgG . The specificity of these antibodies has been determined in previous studies . The tist-test or paired t-test, or one-way or two-way ANOVA with Bonferroni post hoc test for multiple comparisons, as appropriate. When the test for normality failed, the Mann-Whitney rank sum test was applied. Data were considered statistically significant when p < 0.05.All data were expressed as the mean ± SEM. Statistical significance was evaluated using the Student’s 2+ influx in urothelial cells. In the current study, ALI stimulation also evoked a remarkable increase in [Ca2+]i in the cultured urothelial cells , an unspecific MSC blocker, was applied between the two ALI. The GdCl3 (50 μM) treatment almost abolished the second ALI-evoked [Ca2+]i increase  channel , the ENaC channel antagonist, or GsMTX4 , one commonly used Piezo channel antagonist , the specific TRPV4 antagonist, was applied between the two inductions of the ALI and during the second ALI. The amplitude of the second ALI-induced [Ca2+]i increase was reduced by 38.8% , a nonspecific inhibitor of many cation channels, including the TRP channels, significantly reduced the ALI-induced i increase was significantly reduced by 22%, compared with that in the presence of normal HBSS  calcium stores, thapsigargin (Sigma), a blocker that irreversibly inhibits the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pumps that are responsible for calcium reuptake from the cytoplasm back into the ER, was applied between the two ALI stimuli and during the second ALI stimulus. The second ALI-induced [Ca2+]i increase was significantly reduced by 62.4% in the presence of thapsigargin , a membrane-permeable form of BAPTA was applied between the two ALI stimuli and during the second stimulus to clamp intracellular Ca2+ levels (2+]i was significantly reduced (The ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor IP3R;  are the  Xest-C; ; Sigma, increase ,Bb, and increase ,Be. To f+ levels . Second  reduced ,Bf.2+]i increase and release of ATP, 10panx (10panx (10 μM) significantly reduced the second ALI-induced [Ca2+]i increase by 39.3%  to degrade extracellular ATP by catalyzing its breakdown to ADP. As shown in 2+]i increase by 59.7% . Furthermore, PPADS (10 μM) significantly blocked the ALI-induced [Ca2+]i increase by 49.7%  Both Ca2+ influx and Ca2+ release from intracellular Ca2+ stores contribute to the [Ca2+]i increase; (2) TRPV4 or pannexin 1 channels are the two membrane-bound MSCs involved in Ca2+ influx; (3) activation of both IP3 and ryanodine receptors on the ER contribute to Ca2+ release from intracellular Ca2+ stores; and (4) ATP release also contributes to the [Ca2+]i increases by activating P2X and P2Y receptors via autocrine or paracrine mechanisms.In the current study, the cellular mechanisms underlying ALI-induced increase in i increase invalidates the involvement of these MSCs.In previous studies, TRPV4 , epithel+ ENa; , Piezo1 + ENa; , Piezo 2+ ENa; , connexi+ ENa; , and pan+ ENa;  have beeThese results are in contrast with those of previous reports, which state that Piezo1, Piezo2 , ENaC, o10panx, the specific pannexin 1 channel antagonist, significantly attenuated both the ALI-induced [Ca2+]i increase (by 39.3%) and ATP release indicates the involvement of pannexin 1 channels. Furthermore, our immunohistochemical analysis demonstrated the expression of the pannexin 1 channel in urothelial cells.Pannexin 1 channels are characterized by their mechanosensitivity and their ability to form large pores that are permeable to ATP and other molecules . Therefo2+]i increase was reduced by 22% in Ca2+-free HBSS and was reduced by 62.4% following the application of thapsigargin, a blocker that irreversibly inhibits the ER Ca2+-ATPase pumps. These results suggest that compared with Ca2+ entry, Ca2+ release from the ER calcium stores are the dominant source of the ALI-induced [Ca2+]i increase. The contribution of Ca2+ release from the ER calcium stores can also be supported by the observation that ALI induced [Ca2+]i was significantly reduced by application of BAPTA-AM to clamp intracellular Ca2+ levels  the ALI-induced [Ca2+]i increase was significantly reduced by ATP hydrolysis with apyrase and (2) the [Ca2+]i increase was significantly reduced by blocking the P2X and P2Y purinergic receptors with PPADS. Thus, autocrine ATP signaling works like an amplifier to increase the concentration of cytosolic Ca2+. We did not examine which type of purinergic receptors is involved with these processes. However, previous studies have shown that the P2X receptor that mediates the increase in [Ca2+]i is dependent on extracellular Ca2+. On the other hand, P2Y receptors are G-protein-coupled receptors that can induce the release of Ca2+ from intracellular stores via the IP3 pathway , we propose that P2Y receptors may have a greater contribution to the process than P2X receptors.One report on a human urothelial cell line (T24 cells) showed that ATP released upon cell stretching contributes to a major part of Caignaling . In agre pathway . Based o2+ influx, which in turn activates IP3 or ryanodine receptors on the ER to induce Ca2+ release from intracellular Ca2+ stores to further increase [Ca2+]i. Activation of the pannexin 1 channels or [Ca2+]i increase leads to ATP release through nonvesicular or vesicular mechanisms, respectively, which further enhances the increase in [Ca2+]i by activating at P2X and P2Y receptors via autocrine or paracrine mechanisms.Based on the current results, a working model has been proposed . The proNotably, the method used to create the ALI-induced plasma membrane deformation by causing changes in the perfusion volume may alter the equilibrium and concentration of signaling molecules close to the plasma membrane, thereby altering their autocrine/paracrine effects. Typically, this is the case with the ATP concentration; transient reduction of the perfusion fluid volume may increase the concentration of the nucleotide. Compared with other techniques, this is a disadvantage of the ALI method to induce urothelial cell deformation and is a limitation of the current study. Another limitation is the pharmacological approach employed for most of our experiments to determine the associated channels or receptors. Some of the antagonists used in the current study may not be very specific.2+]i increase in urothelial cells. Changes of any one of the associated channels or receptors A-0027].XZ and SW participated in research design. JW, ZC, SW, and MZ conducted experiments. JW, XZ, SZu, ZC, and MZ performed data analysis. XZ, SZh, and JW wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.