Differentiation (1984) 27: 126132 Differen tiat ion 8 Springer-Verlag 1984

Human sperm swimming in flow Siddhartha Sarkar Division of Medical Genetics, Department of Medicine (M-013), University of California at San Diego, La Jolkd, CA 92093, USA

Abstract. To study the movement of human sperm, we have developed a microflow cell by miniaturizing our design for a preparative fractionation flow column. The microflow cell enabled us to view the movement of sperm over periods as long as 2 min. Sequential steps of filming, editing, and analysis revealed that the curved swimming patterns of sperm swimming in stagnant fluid become nearly straight tracks when the flow velocity is increased. However, the net swimming speed remained unchanged. Motile sperm accumulated near solid wall surfaces surrounding the fluid and oriented against the direction of the current; the veloci- ty gradient was steepest in these regions. A laminar-flow preparative column separated motile sperm from dead sperm by carrying the nonmotile sperm and debris with the stream while leaving the motile sperm near the sur- rounding walls.

Introduction Sperm motility and capacitation are essential for migration and fertilization [6], but only a small number of deposited sperm reach the fimbrial end of the oviduct where fertiliza- tion occurs [3, 41, which indicates that the oviduct milieu provides stringent selective conditions for the wide range of variation in sperm population [7, 171. The experiments described here were designed to study the swimming behav- ior of human sperm over a period of several seconds, there- by more closely approaching physiologic reality than the instantaneous swimming response that occurs when sperm are exposed to experimental flow perturbations. Hence, this study is a sequel to work of Adolphi [l], who formulated the relatively long-term effects of flow-induced orientation, and Rothschild, who understood the importance of local velocity gradient [12] and surrounding vessel walls in pro- ducing these effects [13].

Sperm have a tendency to congregate near walls [13] and orient themselves against the direction of moving fluid [ l , 101. Rothschild [12] found that the orientation of sperm is sensitive to local flow velocity gradient, especially near walls where the gradient is the steepest. The locomotion of these slender bodies swimming near vessel walls or be- tween solid boundaries is greatly influenced by their body geometry, the properties of surrounding fluid, and the direc- tion of movement with respect to the wall [2, 51. These effects are complex and behaviorly significant, since sperm

in their natural environment travel through convoluted lu- mens and ducts filled with streaming viscous exudates [7].

Our interest in human sperm swimming was prompted by the need to find a relatively easy and rapid procedure for separating dead sperm and debris from living sperm in human semen [16]. We designed a flow cell to enhance interactions between sperm and a horizontal velocity gra- dient, and also to make use of the fact that sperm released in the path of laminar flow swim toward nearby walls, while the dead sperm are drawn to the center of the parabolic flow.

Our model for the mechanism of sperm separation in a horizontal velocity gradient is illustrated in Fig. 1. Numbers alongside the hollow cylinders (side view) repre- sents a flow-velocity scale of one to ten from the wall to the center of the parbolic profile. Concentric circles (top view) indicate the velocity regions. Sperm inoculated at the center (zone 10) are fractionated in three dimensions by swimming radially outward, as indicated by the arrows (top

st Y

v(r) = a(l - 11) ':

Fig. 1. Schematic model of sperm fractionation by parabolic profile of laminar flow. The relationship between the velocity at any radial point r, the average velocity r, and the radius of the cylinder r,, are calculated by the formula at the lower feft

127

view), and simultaneously upward (side view). Therefore, at any given time, the radial dispersion pattern of sperm across the flow path and in a longitudinal direction is deter- mined by the sperm-velocity vector and the flow-velocity profile. The longer the swimming time required to reach the wall, the more quickly sperm move to the upper frac- tions. Because dead sperm cannot leave the central core of the flow, they accumulate in the top fraction.

Based on this model, we made a cylindrically shaped flow column to separate dead from living sperm. As ex- pected, the dead sperm were concentrated in the top frac- tion. However, to our surprise, motile sperm did not accu- mulate where the flow entered the chamber but were distrib- uted along the entire flowpath. In order to understand the significance of this unexpected distribution of motile sperm, we constructed a microflow cell for observing sperm during prolonged swimming in the presence of flow.

Methods

Semen donors. Semen was obtained from eight fertile volun- teers who were 2741 years old. Sperm counts ranged from 5 x 107/ml to 2 x 108/ml and motility from 30%-80%. Se- men was fractionated within 2 h of collection.

Flow medium. Semen was diluted with two volumes of warm growth medium RPMI 1640 (Gibco) supplemented with 0.5% fetal calf serum, which was preheated to 56" C for 30 min to remove spermicidal factors, and 1 mg/l00ml fructose. The diluted semen was filtered through a 100-pm mesh nylon filter to remove clumps and clots, and then centrifuged for 5 min. The resulting pellet was then resus- pended in 1 ml growth medium containing 1.9% or 1.4% w/v Ficoll 400 (Pharmacia).

Microflow cell. The microflow cell was a plastic chamber (30 x 80 x 5 mm) grooved in both flat sides to fit glass cov- erslips. The medium was fed through two inlets (internal diameter 0.88 mm) set 3.5 mm apart, merged into a central channel (1.1 x 33 x 1.1 mm), and exited through a tube at the opposite side. Coverslips glued (Dow-Corning high-vac- uum silicone lubricant) to the flat sides sealed the chamber, and a metal frame screwed to one side added rigidity.

Cinematographic recording and editing for analyses of swim- ming tracks. Cinemicrophotography was done with a 16-mm Photosonic camera (model 16-1P) at 16 frames/s (calibrated with an internal timing light) with a synchro- nous strobe (Chadwick-Helmuth, model 136) and an expo- sure time of 55 ps. Kodak 16-mm TriX reversal film (Kodak 7278) was used. The movie film was spliced in 8-second-long segments of sperm swimming. Each segment was projected on a screen and photographed with long exposure on a single frame of a 35-mm single-lens reflex (SLR) camera loaded with Kodak Panatomic film (ASA 32). The tracks were printed on a positive film to appear as dark lines on a transparent background. This positive film carrying all the 8-second-long track images was projected frame by frame, and the tracks were traced on a paper. The tracks thus accumulated were retraced with a pen on a graphic tablet of an Apple computer programmed to obtain swim- ming parameters. The magnification scale in all these steps was calibrated by a micrometer, which was first photo- graphed with the sperm by the 16-mm Photosonic camera,

FLOW COLUMN

lml

m

Fig. 2. Cylindrical flow column with the principal components of laminar-flow fractionation technique

and the scale was carried through in all subsequent steps. A phase-I low-magnification Zeiss 10 x objective (ma., 0.22) and a phase-3 Zeiss condenser were used to obtain a microscopic field of sufficient depth for keeping entire lengths of swimming sperm in view.

ReSultS

Preparative fractionation flow column to separate dead sperm from motile sperm

A cylindrical flow column (internal diameter 0.8 cmx height 30 m) made of a 10-ml disposable pipette (Fig. 2) was placed vertically to equalize the effect of sedimentation throughout the chamber and to generate a horizontal veloc- ity gradient [lo, 11, 191. The nutrient flow medium, which contained added Ficoll to increase the viscosity (q 20'2.0 cp for 1.4% solution) and density (p 20'1.005), supported sperm survival for at least 8 h. A velocity between 100 and 150 pm/s (Harvard infusion pump, model 975) was optimal for fractionation. The semen was diluted twofold with the medium, filtered to remove clots, centrifuged to a pellet, and finally resuspended in 1 ml medium of slightly higher viscosity (Ficoll 1.9%, q 20'2.1 cp). The number of sperm in the final suspension ranged from 5 to 20 x 107/ml. The flow column was housed in a controlled environment with an ambient temperature of 35" C. Sperm suspension was injected for 1 min through the central nozzle of the pipette cap, followed by 0.5 ml medium while the pump was run- ning. The pump delivered a total of 0.4 ml/min through the three side inlets to generate an average flow velocity of 120 pm/s in the flow column which filled in 30 min. Sperm fractions were collected with syringe through a hole at each fractionation point, starting at the top. A rubber sleeve lubricated with glycerol which sealed these holes dur- ing the flow was slightly turned after sample withdrawal to reseal the orifices.

The fractionation and calculation of the results are given in the inset of Fig. 3. Also, in Fig. 3 are the distribution of total sperm count and percentage of motile sperm for each fraction listed from the bottom (left) to the top (right). Most of the motile cells were in the lower half, but dead

128

I I - MICROFLOW CELL

" 1 5 10

FRACTION NUMBER Fig. 3. The distribution of sperm swimming speeds (km/s) in each fraction of the flow column. Class 1 or 2 refers to fast and slow sperm, respectively. Numbers in parentheses indicate tracks ana- lyzed in each fraction. Distribution of sperm numbers and percent- age of motile sperm in serial fractions obtained from the bottom (frfi) to thc top (right)

sperm were carried to the upper portion. Although dead sperm contaminated each fraction to some degree, increas- ing the ratio of column capacity to sample volume made possible a 100% yield of motile sperm in the bottom frac- tions (data not shown). The lowest fraction contained most of the debris, such as crystals and precipitates, and the uppermost fraction contained other common contaminants such as small round and large, flat epithelial cells along with dead sperm.

Variations in forward swimming speeds of the fraction- ated sperm are also shown in Fig. 3. The average speed measurements given in the abscissa represent individual sperm's track lengths in microns produced by 1 s swimming [9]. Care was taken to maintain standard assay condition for all fractions. A 50-pm-thick mylar O-ring was placed between the siliconized slide and coverslip and the space was filled with 10 pl sperm suspension from each fraction. A number of tracks (46-180) were used to derive the mean and standard deviation and the number for each fraction is given in parenthesis (Fig. 3).

Sperm in the bottom fractions (class 1) were the fastest swimmers compared to all other fractions (class 2), but standard deviations in all fractions were high (Fig. 3). On average, class 1 cells wam 50% faster (-30 pm/s) than those in class 2 (- 20 pm/s). and their preferential concen- tration at the bottom fractions confirmed the selectivity of the fractionation procedure. However, it was clear that the extent of heterogeneity in speed could not be responsible for the wide distribution of motile sperm among the flow- column fractions (Fig. 3).

Microflow cell to track swimming behavior

By developing a microflow cell (Fig. 4) as a miniature ver- sion of the preparative flow column, we could observe and record sperm movement on motion-picture film. The flow medium was pumped through the unit's two inlets, and sperm suspension was injected through the center inlet into the flow channel. The microflow cell was placed upright on the microscope stage of a Reichert inverted microscope by tilting it to rest on its back, thereby generating a horizon-

mTEAlYMLHAw3ER Erh mltnlla olihca 0.8 ntnl hnmamdirtm 3.5 mm

Fig. 4. Design of the microflow cell used to visualize and record sperm swimming during varying flow conditions

COVER SLIDE (No. 1) 25 x 75 mm

110 :

Fig. 5. Map of horizontal velocity profile in the microflow cell

tal velocity gradient. A calibrating device was attached in place of the focusing knob to record depth in microns. Two siliconized coverslips sealed the front and back walls of the microflow cell's flow channel, which allowed viewing of the entire depth. The width of the channel (1,100 pm) was equal to the width of the field of view of the recording 16-mm camera. The camera operated at 16 frames/s syn- chronous with a strobe. A low magnification objective lens (n.a., 0.22) and a dark field-like illumination provided SUE- cient depth to keep entire lengths of swimming sperm in view.

The velocity profile of the medium's depth (1,100 pm) was measured in the microflow cell at an average flow rate of 156 pm/s (Fig. 5). This flow map was obtained by cine- matographically recording the transit velocities of neutrally buoyant beads (- 2 p) at measured depths. Recordings were made at the midpoint of the well between two long (33 mm) side walls, although the flow profile was slightly off center, tilting toward the back wall. This asymmetry was caused by the two coverslips that were not exactly parallel, due to the uneven layers of lubricant that sealed them. Flow rates could not be measured at the edges because beads

129

Fig.6a-f. Cinematography of swimming sperm. Panels indicate steps involved in recording sperm behavior during static (no flow; a, c, e) conditions and in laminar flow @, d, r). The two 16-mm frames (exposure 1/16s) show sperm orientation in no flow (a) and flow (b); the tracks produced by 8-s-long swimming in no flow (c) and flow (a), and their respective tracings (e, r) used for graphic analyses

rarely traversed these areas. The gradient estimated by ex- trapolation of the observed data was between 0 and 11.8 pm/s within 20 pm of the front coverslip surface and, in the back, it was twice as steep. Even the fastest bead at the central core of the flow took between 2 to 3 s to traverse the 1,100 pm x 850 pm field of the camera. Point 0 in Fig. 5 was the geometric center of the chamber, but the center of the flow was 120 pm toward the back from this point. The velocity profile was symmetrical when mea-

sured from side to side. A Vanguard motion analyzer was used for constructing the map from the 16-mm movie.

The swimming of sperm was recorded in this chamber under the flow conditions already described, in an ambient temperature between 20" and 22" C. All initial recordings were videotaped and analyzed with a video analyzer. Subse- quently, fresh recordings were made on 16-mm film, and the following results were derived from the 16-mm film recording.

130

Table 1. Swimming behavior of sperm in stationary fluid Sperm swimming inflow

Sperm (105/100 pl Ficoll medium) were injected through the central inlet into the microflow cell with the pump shut off. A strong flow generated by injection caused momentary streaming of the sperm along the flow axis which ceased when the flow stopped. Within 2-5 min of injection, a p proximately 98% of motile sperm crowded near the front and back walls and were filmed to record 2 min swimming when there is no flow. After 2 min filming, the pump was started and the microscope was focused on the center of the flow to determine the maximum flow velocity. Since the beads were in the medium at all times, their transit times across the 850-pm-long window of the camera were taken as a measure of maximum flow velocity. After I min pumping, the flow velocity reached a steady state. Three minutes after the pumping started, the camera was refo- cused from the flow's center to the coverslip surface, and filming continued for 2 min. The exposure time was 55 ps and the frame rate was 16 frames/s. The sperm's position and direction reproduced in Fig. 6a and b signify the perva- sive effect of the flow on sperm. The random direction and position without flow (Fig. 6a), compared to upstream orientation during flow conditions (Fig. 6b), should be no- ticed.

A series of editing steps were taken to obtain swimming parameters from the movie recordings. Segments covering a sequence of 128 frames, equivalent to an 8-s span of real swimming time, were spliced. After the first 2 s had elapsed (32 frames), that section was separated. Then a sequence of 1 s (16 frames) was deleted. The deleted gap was replaced (16 frames) with a black leader. In the entire 8-s sequence, sperm swam for 2 s, interrupted with a black lead for 1 s, and then swimming reappeared and continued for another 5 s. The 128-frame-long piece was projected on a screen and rephotographed (time lapse) on a single frame of a 35-mm SLR camera. The single 35-mm frame recorded the distances that spcrm covered in 8 s, with a precise 1 -s interruption between the second and third sec- ond. The 1-s interruption in every track served two pur- poses in analyses: to indicate the direction of swimming since the gap was always at the beginning of the track, and to calibrate swimming speed (wide for fast sperm and hardly visible for slow sperm). The direction was further confirmed by comparing the original 16-mm movie frames with the 35-mm frame (Fig. 6c, d).

Several 35-mm frames of 8-s-long track pictures were traced by projecting all of them onto a single paper (Fig. 6e, f) to accumualte the legible traces, which were retraced with a light pen on a graphic tablet of an Apple computer. Each trace was identified with a number and was analyzed to obtain swimming parameters in a digitized form. Each track was analyzed for average speed, angular velocity, clockwise or counterclockwise (-) movement, radius of curvature, and orientation with respect to the flow axis (Tables 1, 2).

In accordance with the heterogeneity in sperm swim- ming speed in the presence or absence of flow (Fig. 6e, f), the calculated values of coefficient of variation (Table 1, column 2) also indicate a scattered distribution of very long and extremely short tracks, e.g., tracks 19, 29, and 2 in contrast to tracks 28 or 30 (Fig. 6e). The extent of hetero- geneity (Tables 1,2) in swimming speed was nearly the same without flow (SD 8.5) and during flow (SD 8.42). The aver- age swimming speed of 22.6pm/s in stagnant fluid de-

Track Velocity Angular Radius (pm) Orientation' number (pm/s) velocity' of (degr=)

(radian/s) curvature

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Mean SDd C V d

25 0.028

17 0.049 25 0.01 5

25 0.129

12 -0.016

28 -0.171

13 -0.057 13 -0 23 -0.033 35 -0.134 34 -0.138 34 -0.118 32 -0.157 19 -0 24 -0.137 14 0.100 24 -0.026 33 - 0.093 6 -0

29 0.033 10 -0 20 0.119 25 -0.020 25 -0.096 25 0.090 19 -0.163 16 -0.036 30 -0.147 7 0.010

36 -0.197

22.6 0.077 8.5 0.06

0.38 0.78

902 165 764 160 348 168

1,684 29 163 24 196 144 226 66

65 692 130 263 25 243 91 290 74 20 1 101

87 176 27 143 144 949 148 357 74

17 864 21

162 167 51

1,256 166 255 85 275 33 117 108 444 166 204 49 647 64 181 126

462(26) 92 389 53

0.84 0.58

The negative sign refers to counterclockwise and absence of this sign to clockwise direction of track movement. Only absolute values were taken to calculate the mean and standard deviation Only 26 samples, in which radii of curvature could be deter- mined, were used to calculate the mean and standard deviation

' Upstream direction, 9oo-18O0; downstream direction, 0"-90"; random, 90'; flow axis, 0" SD, standard deviation; CV, coefficient of variation

creased by 11 YO to 19.8 pm/s in flowing medium. Although 7 out of 30 had speeds lower than 35 pm/s before the flow, 8 out of 34 low-speed tracks remained with flow. Hence, the current did not strip slow sperm swimming near the coverslip wall. The flow was efficient only in removing dead sperm near the wall, but it did not discriminate the fast from the slow sperm. This observation is compatible with the large standard deviation in swimming velocities ob- served among the flow-column fractions; sperm with one- half to one-quarter the average speed remained with the motile sperm fractions, while the dead sperm were carried away by the leading edge of the flow (Fig. 3).

The twofold decrease in the sperm's angular velocity and a concomitant increase in their movement's radius of curvature were the most pronounced effects of the fluid in motion. The angular velocity decreased from 0.077 ra- dian/s (no flow) to 0.033 radian/s (flow), as evidenced by the considerable straightening in the track curvature (Fig. 6e, 0. However, the standard deviation remained high

131

Table 2. Swimming behavior of sperm in flowing fluid

Track Velocity Angular Radius (pm) Orientation' number (pm/s) velocity" of (degree)

(radian/s) curvature

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Mean SDd CVd

21 27 19 16 32 27 20 15 19 4 5

31 21 15 30 32 36 20 20 17 20 33 13 22 31 26 12 7 7

18 21 11 15 10 19.8 8.42 0.42

- 0.073 -0.013 -0.013 - 0.048 - 0.01 8 -0.18 -0.018 -0 -0

0.01 -0 -0.102 -0 -0 -0.069

0.05 -0

0.057 - 0.045

-0.037 0.09

-0.01 -0.07 -0.025 -0.03

-

0.07 0.03

-0 - 0.037 -0.083 - 0.039 - 0.01 6 - 0.04

0.033 0.029 0.88

286 2,098 1,446

337 1,831 1,537 1,079

390

301

426 598

353 445

561 351

1,144 350

1,242 872 170 244

495 262 287 896 264

704(26) 531

0.75

168 161 147 164 169 152 166 168 166 107 105 110 165 1 69 57

153 144 162 168 168 151 123 155 118 169 143 100 99 96

157 13

151 167 138 140 36 0.26

The negative sign refers to counterclockwise and absence of this sign to clockwise direction of track movement. Only absolute values were taken to calculate the mean and standard deviation Only 26 samples, where radius of curvature could be determined, are used to calculate the mean and standard deviation Upstream direction, 9O0-180"; downstream direction, O0-9Oo; random, 90"; flow axis, 0" SD, standard deviation; CV, coefficient of variation

(CV 0.88 and 0.78), indicating that the angular velocity of the responding population was as heterogeneous as that of the initial population before flow (Tables 1, 2). Equal numbers of sperm swimming tracks faced up- and down- stream in the absence of flow (Table 1; 15 faced each way out of the total 30), as expected if the orientation was ran- dom and independent of geotaxis. However, with flow (Ta- ble 2), the orientation changed from an average of 92" (no flow) to 140" (flow), and the majority of sperm tracks (32/34) faced upstream (> 90"). The frequency distribution was narrow (CV 0.26) because a higher percentage (70%) of sperm tracks were oriented above the 140" average, i.e., between 140" and 180" (maximum orientation), with only 20% between 90" and 140". The high density of cells in the middle of the chamber floor and the paucity on the

sides (Fig. 6e, f) probably reflected the injection site of sperm at the center of the chamber (Fig. 4).

Disclrssioo

Given the heterogeneity of sperm swimming behavior shown by our results in Tables 1 and 2, it is remarkable that fractionation in a preparative flow column provided a high level of motile sperm enrichment (Fig. 3, inset). How- ever, under steady-state flow conditions when sperm were inoculated into the center of the flow, fractionation took place in three dimensions, radially across the gradient and vertically with the flow (Fig. 1). The correlation of the steepness of velocity gradient to fractionation efficiency could not be tested directly, since the velocity profile could not be mapped in the preparative column.

Our subsequent studies of sperm movement during pro- longed fluid flow contain clues to the probable mechanism of their fractionation (Fig. 1) and the broad distribution of sperm along the flow path (Fig. 3). When we videotaped sperm swimming in a microflow cell (Fig. 4), we found that motile sperm, which were initially facing downstream, swam toward the wall and made a U-turn around the flow axis to orient upstream. During this time (which lasted for about 2 min), the swimming speed and angular velocity ap- peared to have changed before sperm returned to swimming with the initial speed on a straight line track. The radius of curvature increased in proportion to the steepness of the velocity profile near the wall. The initial lag period between the onset of the flow and the move to make the U-turn could be important for fractionation.

In contrast, dead sperm changed their orientation in flow by pivoting on their heads if they happened to be near a wall [12]. Dead sperm at the center of the flow did not traverse the horizontal gradient and, together with those near the wall, were drawn into the leading edge of the flow.

Analyses of the cine frames (Tables 1 , 2) revealed a new dimension in the heterogeneity of swimming behavior in flow and the exquisite selectivity of flow against the dead sperm. Sperm swimming with low (&10pm/s) or high (30 pm/s) speed responded uniformly by upstream orienta- tion to flow and by a dramatic increase in the radius of curvature. However, flowing fluid selected stringently for all motile sperm, probably on the basis of their upstream swimming, in preference to any other swimming parameter. The coenicients of variation for speed, angular velocity, and radius of curvature were high before or after the flow; the values were respectively 0.38, 0.78, and 0.84 in station- ary fluid, and the corresponding values in flow were 0.42, 0.88, and 0.75. In contrast, the CV of frequency distribution in orientation (with respect to flow direction) decreased from 0.58 in stationary fluid to 0.26 in flow, indicating a high degree of selectivity [19].

Although this selectivity adequately explains the remov- al of dead sperm by the flow, the broad distribution of motile sperm in the flow-column fractions requires addi- tional explanation (Fig. 3). We postulate that the instanta- neous response of sperm to the rate of change of velocity as they swim across a gradient toward the vessel wall is a decisive factor in their fractionation. Thus, motile sperm fractions recovered from the bottom and top of a prepara- tory flow column (Fig. 3) must somehow differ in sensitivity

132

to the steepness of the gradient and in response time. The sooner they orient upstream after flow begins, the greater is their chance of being fractionated at the bottom. They should also differ in the intensity of response; a sharper U-turn followed by a larger increase in the radius of curva- ture would effectively separate this population at the bot- tom fractions. Other studies ([15]; S. Sarkar, D.J. Jolly, T. Friedmann and O.W. Jones, submitted for publication) have demonstrated that sperm from bottom and top frac- tions swam similarly in stationary fluid, but had distinct and characteristic responses to flow. They were also geneti- cally distinct; the bottom fractions were enriched for X sperm and the top ones for Y sperm.

Results reported here using the microflow cell are prom- ising for the study of instantaneous response of the cell in switching the bending pattern of the flagellar beat [14]. The microflow cell is also suited for studying cilia-flagella interaction for sperm transport. The side surface could be coated with monolayers of ciliated epithelium beating ac- tively to generate a flow [2, 8, 181. Sperm swimming in such a flow environment would serve as a model for explor- ing the nature and intensity of the oviduct’s influence on sperm selection.

Acknowledgemenfs. 1 thank Drs. H. Winet, T. Y-T Wu, G. Yates and G.W. Schmid-Schoenbein for their moderating influence. in the planning of these experiments; C. Dickey, J. Frisbee and R. Arati for their enthusiasm and able assistance, and Dr. O.W. Jones for his continued support of this project.

References 1. Adolphi H (1905) Die Spermatozoen der Saugetiere schwim-

men gegen den Strom. Anat Anz 26: 549-559 2. Blake JR, Liron N, Aldis GK (1983) Flow patterns around

ciliated microorganisms and in ciliated ducts. J Theor Biol (in press)

3. Blandau RJ, Odor DL (1949) The total number of spermatozoa reaching various segments of the reproductive tract in the fe- male albino rats a t intervals after insemination. Anat Rec 103:9f109

4. Blandau RJ, Brackett B, Brenner RM, Boling JL, Broderson SH, Hammer C, Mastroianni L Jr. (1977) The oviduct. In:

Greep RO (ed) Frontiers in reproduction and fertility control. M.I.T. Press, Cambridge, USA, pp 132-145

5. Brennen C, Winet H (1977) Fluid mechanics of propulsion by cilia and flagella. Ann Rev Fluid Med 9: 339-398

6. Chang MC (1951) Fertilizing capacity of sperm deposited in the fallopian tube. Nature 168 : 697-698

7. Katz DF, Overstreet JW (1980) Mammalian sperm movement in the secretions of the male and female genital tracts. In: Stein- berger E, Steinberger A ( 4 s ) Testicular development, structure and function. Raven Press, New York, pp 481489

8. Nevo AC, Weisman Z, Sade J (1975) Cell proliferation and cell differentiation in tissue cultures of adult mucociliary epi- thelia. Differentiation 3: 79-90

9. Overstreet JW, Katz DF, Hanson FW, Fonesca JR (1979) A simple, inexpensive method for objective assessment of human sperm movement characteristics. Fertil Steril 31 : 162-172

10. Roberts AM (1970) Motion of spermatozoa in fluid streams. Nature 228 : 375-376

11. Roberts AM (1975) The biased random walk and the analyses of microorganism movement. In: Wu TY-T, Brokaw CJ, Bren- nen C (eds) Swimming and flying in nature, vol 1. Plenum Press, New York, pp 377-393

12. Rothschild L (1962) Sperm movement problems and observa- tions. In: Bishop DW (ed) Spermatozoan motility. Am Assoc Adv Sci, Washington Publ. no. 72, pp 13-29

13. Rothschild L (1 963) Nonrandom distribution of bull sperma- tozoa in a drop of sperm suspension. Nature 198: 1221-1222

14. Russo AF, Koshland DE Jr. (1983) Separation of signal trans- duction and adaptation functions of the aspartate receptor in bacterial sensing. Science 220: 101~1020

15. Sarkar S (1981) Human X and Y sperm : a laminar flow method of in vitro separation. Fed Proc: 3037 (abstract)

16. Sarkar S, Jones OW, Centerwall W, Tyler ET, Shioura N (1978) Population heterogeneity in human sperm DNA content. J Med Genet 15:271-276

17. Sobrero AJ (1963) Sperm migration in the female genital tract. In: Hartman CG (ed) Mechanisms concerned with conception. Pergamon, New York, pp 173-203

18. Verdugo P, Johnson NT, Tam P (1980) 8-adrenergic stimula- tion of respiratory ciliary activity. J Appl Physiol48:868-871

19. Walton A (1952) Flow orientation as a possible explanation of ‘wavemotion’ and ‘rheotaxis’ of spermatozoa. J Exp Biol 29 : 52&531

Received November 1983 / Accepted in revised form March 1984