Behavioural Brain Research 136 (2002) 325 /328 www.elsevier.com/locate/bbr
Short communication
Do sperm cells remember? Peter Brugger a,*, Ervin Macas b, Ju¨rgen Ihlemann c a
b
Department of Neurology, University Hospital Zu¨rich, CH-8091 Zu¨rich, Switzerland Department of Obstetrics and Gynecology, University Hospital Zu¨rich, CH-8091 Zu¨rich, Switzerland c Laser Laboratory Go¨ttingen, Go¨ttingen, Germany Received 24 April 2002; received in revised form 13 May 2002; accepted 13 May 2002
Abstract Spontaneous alternation behavior (SAB) is the universal tendency of animals, including unicellular organisms, to alternate directional choices at consecutive left/right branchings while traversing a maze. Occurrence of SAB implies short-term memory, as a current decision is statistically dependent on previous ones. We developed a procedure to assess SAB in human spermatozoa. A total of 1302 progressively motile spermatozoa from healthy donors were observed as they entered one of two mazes, both fabricated by eximer laser ablation. The control maze was a simple T-maze (width /depth /20 mm, distance between entrance and free choice Tintersection /600 mm). The experimental maze was identical to the control maze except for a forced right-turn 600 mm before the Tintersection. We recorded individual sperm cells’ left/right decisions at the T-intersections in both mazes. Of the 714 spermatozoa entering the control maze, 49.1% turned to the left (not significantly different from the chance expectation of 50.0%). Of the 588 spermatozoa entering the experimental maze, 58.6% turned left after the initial forced right turn (significant SAB; P /0.041, Wilcoxon). The statistical dependency of a directional decision on a previous one suggests a physiological ‘memory’ in human spermatozoa. Among the possible underlying mechanisms are refractory processes in structures responsible for flagellar beating, a postulation which deserves further scrutiny with video-monitored single-cell testing. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Spontaneous alternation behavior; Human spermatozoa; Physiological memory; Refractory processes
Spontaneous alternation behavior (SAB) refers to the tendency of an animal to alternate left/right turns at consecutive directional choice points while exploring a maze in the absence of reinforcement [5]. Although mainly demonstrated in rodents, SAB appears to be a universal phenomenon of sequential responding: it occurs in man [17] and in lower invertebrates alike [12], and is even observed in unicellular organisms [16]. This widespread occurrence throughout the animal kingdom suggests that the tendency to alternate has basic biological roots; SAB may enhance an individual’s survival value by facilitating foraging and exploratory behavior and by protecting against perseverative tendencies [7]. This work was presented at the First Joint Meeting of the European Brain and Behavior Society and European Behavioral Pharmacology Society, September 8 /12, 2001, Marseille (France). * Corresponding author. Fax: /41-1255-4429 E-mail address:
[email protected] (P. Brugger).
SAB has long been employed to assess memory in the very short-term range and, particularly in rodents, as an indicator of hippocampal development [6]. While experimentally induced retention deficits typically abolish SAB [14], pharmacological enhancement of memory generally increase alternation tendencies e.g. [18,20]. In small organisms, spontaneous alternation paradigms are especially useful for determining the time course of forgetting, operationalized as systematic changes in the distance between ‘forced’ and ‘free’ turn. This distance can easily be plotted against the observed magnitude of SAB, with rates of alternation typically decreasing with increasing length between consecutive choices. Chance alternation is commonly interpreted as indicating that memory traces of the previous direction turned have faded away [1,9,11]. What follows is a first attempt to observe human spermatozoa’s directional ‘choices’ in a maze with one forced turn. Specifically, our goal was to assessed whether SAB was demonstrable in these organisms.
0166-4328/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 2 ) 0 0 1 2 7 - 4
326
P. Brugger et al. / Behavioural Brain Research 136 (2002) 325 /328
Fig. 1. The MC (left) and the ME (right). Note that the width of both main maze arms measured somewhat less than 20 mm (not drawn to scale!), just allowing the sperm cells to propagate by a natural flagellar amplitude (i.e. 17 mm [4]). Depth of all maze parts also measured 20 mm. Inlet, scanning electron micrograph of the T-intersection of one of the mazes.
Two types of mazes were developed (Fig. 1), a control (MC) and an experimental maze (ME). Both were fabricated in polycarbonate material by excimer laser ablation (wavelength 193 nm). Laser ablation using ultraviolet wavelengths is a versatile method to fabricate micro structures such as wells, holes, channels, gratings, or lens-shaped surfaces [13]. A variable rectangular mask is employed to depict the segments of the maze structure. By illuminating this mask with the laser beam and projecting it on the polycarbonate surface, material in the corresponding area is ablated. The ablation depth is controlled by the number of applied laser pulses. Semen samples were obtained from a total of 11 young and healthy donors. The concentration, motility and morphology of spermatozoa were evaluated according to current World Health Organization (WHO) parameters [21]. For the swim up method, one part of semen was mixed with approximately ten parts medium, and the suspension was centrifuged twice at 200 /g for 5 min. The final pellet was again suspended in 1 ml of medium and stored in an incubator (37 8C) for 1 h in a sterile tube. One part of the motile sperm suspension was taken after that time, and its concentration was adjusted to between 5 and 6 millions of motile spermatozoa per ml with medium before each experiment. The microscope slides whose undersides contained the mazes were placed in petri dishes and covered with a tissue culture medium containing the semen samples. Under a microscope, a laboratory assistant naı¨ve to the tested hypothesis tracked those sperms which swam into the maze. Sperm in MC and ME were observed blockwise and up to maximally 1 h or a total of 100 observations (112 for MC in session 11). Each maze type was used once during one session. If a sperm cell adhered to some rough part of a lateral maze wall for
longer than 10 s ( B/5% of trials) it was excluded from tabulation. The criterion for scoring a turn was that a cell’s entire head entered a terminal compartment. If more than ten cells accumulated in one compartment, a new observation block was started with a new semen sample. This was rarely necessary since the sperm cells could leave the compartment through the medium-filled gap present between the floor of the petri dish and the microscope slide. Similar departures never occurred in the more narrow T-arms. In 11 consecutive sessions, 714 spermatozoa were observed as they entered MC and made a directional choice. There was no systematic preference for one direction over the other, 49.1% of the cells turning to the left and 50.9% to the right (Table 1). In the same eleven sessions, 588 spermatozoa made a directional choice after having entered ME, 58.6% turned to the left, a value significantly above the chance expectation of 50.0%; Wilcoxon Z /2.0, P /0.041 (Table 1). After correction for the percentages of left turns observed in the control maze during the same session, the mean excess rate of left turns in ME was 9.5% (Z /1.96, P / 0.050). The present results raise three pressing questions. (1) Do our data document the occurrence of SAB in spermatozoa to a reasonably convincing degree? (2) Assuming they do, can we infer from these data anything about a ‘memory’ capacity in sperm cells? (3) Again assuming that such an inference is appropriate, which physiological processes possibly underlie this phenomenon? Re. 1. While there were comparable numbers of left and right turns in the control maze, the spermatozoa in the ME (with a forced right turn preceding the ‘free choice’) turned more often to the left. Albeit significant, the rate of SAB (58.6%) was small compared with the rates reported for various animal species (in rodents often /80% [5]). This may mainly be the consequence of the relatively long distance between the forced and ‘spontaneous’ turns (more than ten times the length of a single cell, including the flagellum). Hughes [11], for instance, observed a 75% alternation rate for woodlice when the distance between forced and free choices was about twice to three times the length of the animal. Alternation rate decreased to 50% (i.e. chance) for distances larger than ten times a woodlouse’s body length. Also, an individual cell’s turning direction at the T-intersection may have been influenced, in yet unknown ways, by those cells which had already accumulated in the left and right compartments beyond the point of ‘free choice’. ‘Pack behavior’, possibly mediated by chemosensory sperm-sperm-interactions, has in fact been reported in free-swimming spermatozoa [19]. In future research, video-monitored testing of individual sperm cells (allowing for later off-line analyses of swim
P. Brugger et al. / Behavioural Brain Research 136 (2002) 325 /328
327
Table 1 Number of spermatozoa observed and percentages of left-turns (%L ) for the two maze types and the eleven test sessions separately Session number
1 2 3 4 5 6 7 8 9 10 11 Total Mean S.D.
Number of valid observations in
%L choices in
MC
ME
MC
ME
45 50 50 10 100 100 100 79 25 43 112 714
50 30 30 12 100 100 100 80 23 26 37 588
64.4 62.0 22.0 50.0 52.0 65.0 49.0 49.4 44.0 51.2 31.3
64.0 73.3 36.7 66.7 58.0 58.0 50.0 53.8 78.3 46.2 59.5
0.4 11.3 14.7 16.7 6.0 7.0 1.0 4.4 34.3 5.0 28.2
49.1 12.6
58.6 11.4
9.5 12.6
times and flagellar movement parameters) should replace the ‘mass-tests’ described in the present report. Re. 2. Besides information storage, the only other theoretical possibility for the observed above-chance alternation is one involving thigmotaxis, i.e. the tendency of an organism to maintain contact with the walls of a compartment. According to this account, spermatozoa would simply continue to follow the left border of the maze arm after the forced right turn. Thus, the probability of later turning to the left at the point of the ‘free choice’ would slightly increase. Although some evidence suggests that spermatozoa prefer to crawl alongside walls at low population densities [10], thigmotaxis has been ruled out as a main factor involved in the SAB of lower invertebrates, including unicellular organisms [8,15]. It thus appears that the direction of the free choice in the experimental but not in the control maze is at least in part determined by the direction of the preceding turn. Hence, single sperm cells somehow encode information about movement direction in short-term ‘memory’. Re. 3. One candidate mechanism for this information storage entails refractory processes in cell structures responsible for flagellar beating [4]. A forced turn to one side would result in a functional asymmetry in flagellar beating, the compensation of which would impose a bias to turn to the opposite side at subsequent turn points. We propose then, that sperm cells do in fact remember the direction of a previous turn, not using a proper memory system, but rather by means of a fatigue effect in the bind/relax cycling mechanism responsible for flagellar activation [3]. In arthropode species, similar asymmetric fatigue effects in the peripheral motor system have been implicated in the genesis of SAB [2].
%LME %LMC
Acknowledgements We greatly acknowledge Natalia Ka¨lin’s (Zu¨rich) admirable patience during data collection. We thank Thomas Bak (Cambridge) for stimulating discussions and Gerd Marowsky (Go¨ttingen) for introducing us to the technique of excimer laser ablation. We also thank Bruno Imthurn (Zu¨rich), Stefan Puschmann (Go¨ttingen) and especially Marinella Rossetti (Zu¨rich) for excellent methodological advice. Finally, Kirsten I. Taylor’s (Zu¨rich) editorial help is greatly appreciated.
References [1] Akre RD. Correcting behavior by insects on vertical and horizontal mazes. Kansas Entomol Soc 1964;37:169 /86. [2] Beale IL, Webster DM. The relevance of leg-movement cues to turn alternation in woodlice (Porcellio scaber ). Anim Behav 1971;19:353 /6. [3] Cosson J. A moving image of flagella: news and views on the mechanisms involved in axonemal beating. Cell Biol Int 1996;20:83 /94. [4] David G, Serres C, Jouannet P. Kinematics of human spermatozoa. Gamete Res 1981;4:83 /95. [5] Dember WN, Richman CL. Spontaneous alternation behavior. New York: Springer, 1989. [6] Douglas RJ. The development of hippocampal function: implications for theory and therapy. In: Isaacson RL, Pribram KH, editors. The hippocampus: neurophysiology and behavior, vol. 2. New York: Plenum Press, 1975:327 /61. [7] Douglas RJ. Spontaneous alternation behavior and the brain. In: Dember WN, Richman CL, editors. Spontaneous alternation behavior. New York: Springer, 1989:73 /108. [8] Grosslight JH, Harrison PC. Variability of response in a determined turning sequence in the meal worm (Tenebrio molitor ): an experimental test of alternative hypotheses. Anim Behav 1961;9:100 /3.
328
P. Brugger et al. / Behavioural Brain Research 136 (2002) 325 /328
[9] Grosslight JH, Ticknor W. Variability and reactive inhibition in the meal worm as a function of determined turning sequence. J Comp Physiol Psychol 1953;46:35 /8. [10] Hossain AM, Barik S, Rizk B, Kulkarni PM, Thorneycroft IH. Analysis of in vivo migration patterns of human spermatozoa by a petri dish-based horizontal column. Biol Reprod 1999;61:406 / 10. [11] Hughes RN. Turn alternation in woodlice (Porcellio scaber ). Anim Behav 1967;15:282 /6. [12] Hughes RN. Phylogenetic comparisons. In: Dember WN, Richman CL, editors. Spontaneous alternation behavior. New York: Springer, 1989:39 /57. [13] Ihlemann J, Wolff-Rottke B. Excimer laser micro machining of inorganic dielectrics. Appl Surf Sci 1996;106:282 /6. [14] Isseroff A. Limited recovery of spontaneous alternation following hippocampal damage: evidence for a memory impairment. Exp Neurol 1979;64:284 /94. [15] Kupfermann I. Turn alternation in the pill bug (Armadillidium vulgare ). Anim Behav 1966;14:68 /72.
[16] Lepley WM, Rice GE. Behavior variability in paramecia as a function of guided act sequences. J Comp Physiol Psychol 1952;45:283 /6. [17] Pate JL, Bell GL. Alternation behavior of children in a crossmaze. Psychonom Sci 1971;23:431 /2. [18] Ragozzino ME, Unick KE, Gold PE. Hippocampal acetylcholine release during memory testing in rats: augmentation by glucose. Proc Natl Acad Sci USA 1996;93:4693 /8. [19] Sepsenwol S, Taft SJ. In vitro induction of crawling in the amoeboid sperm of the nematode Ascaris suum . Cell Motil Cytoskeleton 1990;15:99 /110. [20] Stone WS, Walser B, Gold SD, Gold PE. Scopolamine and morphine-induced impairments of spontaneous alternation performance in mice: reversal with glucose and with cholinergic and adrenergic agonists. Behav Neurosci 1991;105:264 /71. [21] World Health Organization. WHO laboratory manual for the examination of human semen and sperm /cervical mucus interaction 3rd ed. Cambridge: Cambridge University Press, 1992.
