Human Reproduction, Vol.30, No.4 pp. 884–892, 2015 Advanced Access publication on January 21, 2015 doi:10.1093/humrep/dev002 ORIGINAL ARTICLE Reproductive biology Behavioral mechanism of human sperm in thermotaxis: a role for hyperactivation Sergii Boryshpolets, Serafı´nPe´rez-Cerezales, and Michael Eisenbach* Downloaded from https://academic.oup.com/humrep/article/30/4/884/613882 by guest on 29 August 2020 Department of Biological Chemistry, Weizmann Institute of Science, 7610001 Rehovot, Israel *Correspondence address. E-mail: [email protected] Submitted on September 23, 2014; resubmitted on December 2, 2014; accepted on January 2, 2015 studyquestion: What is the behavioral mechanism underlying the response of human spermatozoa to a temperature gradient in thermo- taxis? summary answer: Human spermatozoa swim up a temperature gradient by modulating their speed and frequencies of hyperactivation events and turns. what is known already: Capacitatedhuman spermatozoa are capable of thermotactically responding to atemperature gradient with an outcome of swimming up the gradient. This response occurs even when the gradient is very shallow. study design, size, duration: Human sperm samples were exposed to a fast temperature change. A quantitative analysis of sperm motility parameters, flagellar wave propagation, and directional changes before, during, and after the temperature change was carried out. participants/materials, setting, methods: The swimming behavior of 44 human sperm samples from nine healthy donors was recorded under a phase-contrast microscope at 75 and 2000 frames/s. A temperature shift was achieved by using a thermoregulated micro- scope stage. The tracks made by the cells were analyzed by a homemade computerized motion analysis system and ImageJ software. main results and the role of chance: A temperature shift from 31 to 378C resulted in enhanced speed and a lower fre- quency of turning events. These were reflected in a 35 + 1% (mean + SEM) increase of the straight-line velocity, 33 + 1% increase of the average path velocity, 11 + 1% increase of the curvilinear velocity, 20 + 1% increase of the wobble, and 4 + 1% increase of the linearity. Qualitatively, the inverse trend was observed in response to a 37-to-318C shift. In addition, the amplitude of flagellar waves increased close to the sperm head, resulting in higher side-to-side motion of the head and, often, hyperactivation. This increase in the extent of sperm hyperactivation was reflected in an increase in the average (mean + SEM) fractal dimension from 1.15 + 0.01 to 1.29 + 0.01 and inthepercentageofhyperactivatedspermatozoafrom3+ 1% to 19 + 2%. These changes in hyperactivation were observed less often in sperm populations that had not been incubated for capacitation. All these changes partially adapted within 3–10 min, meaning that following the initial change and while being kept at the new temperature, the values of the measured motility parameters slowly and partially returned toward the original values. These results led us to conclude that spermatozoa direct their swimming in atemperature gradient by modulating the frequency of turns (both abrupt turns as in hyperactivation events and subtle turns) and speed in a way that favors swimming in the dir- ection of the gradient. limitations, reasons for caution: The conclusions were made on the basis of results obtained in temporal and steep tempera- ture gradients. The conclusions for spatial, shallow gradients were made by extrapolation. wider implications of the findings: This is the first study that reveals the behavior of human spermatozoa in thermotaxis. This behavior is very similar to that observed during human sperm chemotaxis, suggesting commonality of guidance mechanisms in mammalian spermatozoa. This study further substantiates the function of hyperactivation as a means to direct spermatozoa in guidance mechanisms. study funding/competing interest(s): The authors have no conflict of interest and no funding to declare. Key words: human sperm thermotaxis / sperm motility / sperm guidance / sperm flagella & The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected] Behavioral mechanism of human sperm in thermotaxis 885 Introduction hard disk in AVI format. Analyses of the sperm recordings were performed by ImageJ software and a computer-assisted sperm analyzer (CASA) plugin Mammalianspermatozoamustbe guided in order to reach theoocyte(Eisen- (Wilson-Leedy and Ingermann, 2007) upgraded according to Purchase and bach and Giojalas, 2006). Indeed, human spermatozoa were found capable Earle (2012). The analysis was carried out second by second, with 75 frames of responding to chemoattractant gradients by chemotaxis (Ralt et al., 1994), in each second. The following parameters were measured and used in this to a temperature gradient by thermotaxis (Bahat et al.,2003), and to fluid study: curvilinear velocity (VCL, time-averaged velocity of a sperm head m flow by rheotaxis (Miki and Clapham, 2013), all three being potential guid- along its actual curvilinear path, expressed in m/s), average path velocity (VAP, velocity over an average path generated by a roaming average; mm/s), ance mechanisms. On the basis of the sperm response to a concentration straight-line velocity (VSL, the time-average velocity of the sperm head along jump of a chemoattractant, the behavioral mechanism by which capacitated a straight line from its first position to its last position; mm/s), linearity (LIN, spermatozoa respond to the gradient in chemotaxis was suggested to involve defined as VSL/VAP), and wobble (WOB, defined as VAP/VCL). Two para- modulation of the frequency of hyperactivation events and turns in response meters were used for identifying hyperactivation, employing a homemade to the gradient: Reduction when swimming up the gradient and elevation computerizedmotionanalysissystem inMatLab,based onprinciplesdescribed when swimming down the gradient (Armon and Eisenbach, 2011). by Crocker and Grier (1996): the fractal dimension (FD) and the percentage of Downloaded from https://academic.oup.com/humrep/article/30/4/884/613882 by guest on 29 August 2020 A behavioral mechanism for rheotaxis has also been proposed (Miki hyperactivated spermatozoa. FD is an expression of the degree to which the and Clapham, 2013; Kantsler et al., 2014). In this mechanism, too, hyper- sperm trajectory fillsaplane (Mortimeretal.,1996).Ifthe trajectory isa straight activation appears to be involved, assisting spermatozoa to adjust their linewith nodeviations, its FDvalue is1.0 becauseit isonlyinthe first dimension motility according to the fluid flow (Miki and Clapham, 2013). In contrast (length). If, however, the trajectory is meandering, as in the case of a hyperac- to chemotaxis and rheotaxis, there is no information about the behavior- tivated spermatozoon, it covers more of the plane and it has a value closer to 2.0. The FD values of most of the spermatozoa in a microscope field are al mechanism of thermotaxis.Uncovering this mechanism is of special im- between 1.0 and 2.0, although a few spermatozoa may have values larger portance in view of the sperm ability to respond by thermotaxis even to than 2.0 (Katz and George, 1985). FD is thus used as a measure of the intensity extremely shallow temperature gradients (Bahat et al., 2012). Our aim in of hyperactivation (Mortimer et al.,1996). The percentage of hyperactivated this study was to reveal this mechanism. spermatozoa was determined according to the criterion of spermatozoa having both FD .1.4 and VCL .70 mm/s (Armon and Eisenbach, 2011). Materials and Methods High-speed recording of sperm motility Ethical approval High-speed recordings of sperm motility were carried out at the Laboratory of Reproductive Physiology in Faculty of Fisheries and Protection of Water, The study was approved by the Bioethics and Embryonic Stem Cell Research University of South Bohemia, Czech Republic, using a high-speed video Oversight Committee of the Weizmann Institute of Science. Human semen camera (Olympus i-speed TR, Japan, providing 848 × 688 pixels spatial reso- samples wereobtained from healthy donors after3 days of sexual abstinence. lution) at 2000 frames/s, mounted on an inverted microscope (Olympus Informed consent was obtained from each donor. IX83, Japan). Recordings were initiated immediately after the temperature reached the final value of 378C (for positive 31-to-378C gradient) or 318C Sperm sample preparation (for negative 37-to-318C gradient). Semensampleswithnormalsperm density,motilityandmorphologyaccording to World Health Organization guidelines (2010) were allowed to liquefy for Statistical analysis 30–60 min at room temperature and then separated from the seminal Forty-four human sperm samples from nine donors in total were analyzed in plasma by centrifugation (120×g, 15 min, twice) with Flushing Medium (Med- this study. All the samples were temperature responsive. Datawere analyzed iCult, Jyllinge, Denmark). Subsequently, the sperm concentration was adjusted by Statistika software (StatSoft, Inc., Tulsa, OK, USA). Each experimental to 70 × 106 cells/ml with Flushing Medium supplemented with additional point in the graphs is an average of 10–50 spermatozoa (all the motile cells human serum albumin, bringing its final concentration to 0.3%, and the sperm- seen in the microscope field) measured for 1 s each. No statistical compar- atozoa were incubated