Sperm Maturation in the Male Reproductive Tract : Development of Motility ' PENELOPE GADDUM 2 Department of Biological Structure, University of Washington, Seattle, Washington ABSTRACT Rabbit spermatozoa were removed from various levels of the male reproductive tract. They were examined in Hanks' solution at room temperature with a phase contrast microscope and their motility characteristics were recorded cinematographically. Spermatozoa from the seminiferous tubules and ductuli efferentes show weak, vibratory movements with no forward progress. Little change in motility occurs until the sperm reach the flexure of the caput epididymidis where some are capable of moving more vigorously in a circular fashion. Samples from the distal caput epididymidis show a sudden increase in sperm activity and a consistent pattern of tight, circular movement. As the sperm traverse the corpus epididymidis, increasing numbers show progressive, forward movement with longitudinal rotation. The proportion of such sperm becomes significant only in samples from the upper cauda epididymidis and more distal regions. Sperm from the ductus deferens rarely retain the circular movement. It is concluded that rabbit spermatozoa undergo a distinct sequence of changes in their swimming movements as they mature in the epididymis. A similar change was noted in epididymal spermatozoa from the rat and guinea pig suggesting that this process i s fundamental to sperm maturation in several species. It is well known that mammalian spermatozoa undergo a change during their passage through the epididymides which enables them to fertilize ova (Young, '31; Bedford, '66; Orgebin-Crist, '67b). This maturation is accompanied by certain physiological and biochemical changes which have been discussed extensively in earlier reviews (Bishop and Walton, '60; Bishop, '61). Morphological alterations in the acrosome have been noted also (Fawcett and Hollenberg, '63; Bedford, '65). Recently Blandau and Rumery ('64) demonstrated that rat spermatozoa taken from the caput epididymidis swim in a circular fashion whereas those taken from the cauda tend to move in straight lines. When caput spermatozoa were inseminated into the cornu they did not go through the utero-tuba1 junction into the oviduct and thus the number of fertilized eggs was negligible. In contrast, spermatozoa from the cauda epididymidis reached the site of fertilization readily. This experiment emphasized that rat spermatozoa must attain a specific pattern of motility before they reach full fertilizing capacity. Preliminary observations indicated that rabbit spermatozoa undergo similar changes in their swimming pattern during maturation ANAT. REC..161: 471-482. (Gaddum, '67). This finding prompted us to study the motility patterns of rabbit spermatozoa in detail as they move through the various segments of the male reproductive tract. MATERIALS AND METHODS Twenty-five mature male rabbits of various breeds weighing between eight and ten pounds were used in the experiment. Each animal was stunned by a blow on the head and bled immediately by severing the carotid arteries. The testes, epididymides and ducti deferentia were removed quickly and placed in Petri dishes. Connective tissue and blood vessels were cut away and the fat pad over the caput epididymidis was dissected with iridectomy scissors to expose the ductuli efferentes and the various components of the caput epididymidis. In a preliminary investigation involving 12 rabbits, spermatozoa were recovered from several levels of the tract and examined in the manner described below. When it had been established beyond doubt that there were consistent variations in the mo1 Supported by a bio-medical fellowship from the Population Council and, by USPHS grant HD-03464 from the National Instltutes of Health awarded to rzl.nA=., Dr. Richard J. YA~..Y..". 2 Population Council Fellow, 1965-1968. 471 472 PENELOPE GADDUM Fig. 1 Drawings of the right testis and epididymis of the rabbit. A. Lateral view, B. Posterior view, C. Anterior view. D. D., ductus deferens. The numbers 1 to 9 refer to segments of the epididymis which were chosen for sampling the sperm population. tility pattern of spermatozoa removed from different levels, an additional 13 rabbits were used for a systematic study. For this experiment, the epididymis was divided arbitrarily into nine segments which could be distinguished macroscopically by differ- ences in the diameter and convolutional pattern of the tubule, and also by their spatial relationship to the testis (fig. 1 ) . These segments were chosen for convenience of sampling rather than to correspond with regional differences in their DEVELOPMENT O F SPERM MOTILITY cellular characteristics. They do not correspond with the segments of the rabbit epididymis described by Nicander (’57). As each segment of the epididymis was separated from the rest of the tissue, it was rinsed thoroughly in Hanks’ solution, placed in the well of a Maximow slide with a few drops of Hanks’ solution and incised carefully with iridectomy scissors. If the spermatozoa were not released in sufficient numbers by this method, the tubules were pressed gently with a fine dental probe to express their contents. In order to examine the spermatozoa, they were mounted in vaseline rings prepared in the following manner. A ring of vaseline slightly smaller than a no. 1 round coverslip (18 mm) was made on the center of a specially cleaned microscope slide. A 2-3 mm gap was left in the circumference of the ring. After a few drops of the sperm suspension had been deposited in the center of the ring, the coverslip was lowered carefully onto the preparation and the gap was then sealed with a drop of mineral oil. The purpose of the vaseline ring was to raise the coverslip approximately 0.5 mm from the surface of the slide and thus ensure sufficient depth in the preparation so that the spermatozoa could display their normal swimming activity. All preparations were maintained at room temperature. Preparations of spermatozoa were made in this way from epididymal segments one to nine (fig. 1); from the seminiferous tubules dissected from the testis; from the ductuli efferentes and from the ductus deferens. In addition, short segments of the ductuli efferentes were mounted intact in vaseline ring preparations in order to examine the activity of cilia lining their walls. Suspensions of spermatozoa from the ductus deferens were obtained by flushing them from the lumen with Hanks’ solution. The preparations were examined in detail with the phase contrast microscope and the movements of the spermatozoa were recorded cinematographically on 16 mm motion picture film at a speed of 24 frames per ~econd.~ 473 OBSERVATIONS tive tract. The following terms will be used to denote the various types of swimming movement which were observed in the in vitro preparations: ( 1) Vibratory movement. The flagellum moves rapidly from side to side in an oscillatory, or quivering motion. This movement does not result in forward progress. For the most part, the spermatozoon remains in the horizontal plane with the flat surface of the head parallel with the surface of the slide. Often the amplitude of movement is so small as to be barely perceptible, but in some cases, the amplitude has increased significantly. Thus, two types of vibratory movement are distinguished, namely “weak and “vigorous.” ( 2 ) Circular movement. The flagellum is bent in the shape of a curve and the spermatozoon moves in a tight circle rather than forward (figs. 1-5). The diameter of the circular orbit appears to be related to the degree of flagellar curvature. The head of a spermatozoon moving in this fashion apparently remains in one plane through many swimming “strokes.” Such sperm may be seen moving in either a horizontal, oblique or perpendicular plane with respect to the glass surface. When many sperm in a preparation show the circular movement, they may swim in either a clockwise or a counterclockwise direction. ( 3 ) Darting movement. The head of the spermatozoon is propelled in an irregular, zigzag fashion. Forward movement is minimal and highly erratic. ( 4 ) “Tuning forh” movement. The spermatozoon moves forward but its path is a wide arc rather than a straight line. The head does not rotate and the flagellum appears to vibrate so rapidly that it resembles the oscillations of a tuning fork. (5) Rotatory movement (fig. 6 ) . Undulations of small amplitude pass down the flagellum swiftly and the whole sperm rotates about its longitudinal axis. As the head rotates, one observes the well-known “flashing” effect which is due to periodic changes in light scattering as the head changes its position. The sperm makes rapid forward progress in a relatively straight line. This appeared to be the most Rabbit spermatozoa display characteristic patterns of behavior when recovered from specific regions of the male reproduc- 3 A research film demonstrating the various types of sperm movement is available upon request from the University of Washington Press, Seattle, Washington 98105. 474 PENELOPE GADDUM efficient form of movement observed in this study and we consider it to be the motility pattern of fully mature spermatozoa. Motility o f sperm f r o m the seminiferous tubules and ductuli efferentes Most sperm from the seminiferous tubules and the ductuli efferentes show weak, vibratory movements. Their flagella are often curved slightly and maintain this state during movement. On very rare occasions, the position of the head changes so that its thin, lateral edge comes into view. This rotation occurs so rarely that it is impossible to be certain whether it is a simple oscillation of the head from side to side or a complete rotation about the longitudinal axis of the sperm. In preparations from the seminiferous tubules, sperm are either free in the medium or attached by their heads to Sertoli cells. The flagellar movements of the attached sperm are more varied than those of the free sperm, for they are capable of wide, sweeping movements as well as restricted vibratory motion. Occasionally, the distal tips of the flagella have small “blebs” on them which appear to be either remnants of cytoplasm or the end-pieces of the flagella in a tightly folded condition. Intracellular flagella in spermatids are capable of whiplash movements which are similar to those observed in preparations of rat spermatids by Austin and Sapsford (’51) and by Blandau (unpublished observations). Whole mounts of portions of the ductuli efferentes reveal the remarkable activity of the cilia lining these ducts. The ductuli are so tortuous that each appears to consist of many distinct compartments. The cilia lining their walls create strong currents which move the contents around in a brisk, circular fashion. On many occasions, the contents of one compartment may be seen moving off gradually into the adjacent one. A further demonstration of the intense ciliary activity may be obtained by slitting the tubules longitudinally and observing the strong currents in the surrounding fluid. Both motile and non-motile spermatozoa which come into contact with the ciliated epithelium are swept briskly over its surface. Motility o f sperm f r o m the epididymis Segment 1 Spermatozoa from segment 1 (fig. 1 ) show similar motility characteristics to those from the seminiferous tubules and ductuli efferentes. An occasional sperm shows vigorous vibratory movements. Only a small number of sperm can be recovered from this segment. Segment 2 Spermatozoa are more numerous in segment 2 (fig. 1 ) and there is a significant increase in their activity. Nearly all show vigorous vibratory movements, although forward progress is still extremely limited. The flagellum in many cases appears slightly curved and stiff. The bending of the flagellum is very pronounced in a few spermatozoa, and they move in a slow, spasmodic, circular fashion. The proportion of sperm moving in this manner vanes between animals. In some cases, only an occasional sperm shows circular movement while in others, approximately one-third move in this fashion. On rare occasions a few spermatozoa show the rotatory movement so typical of fully mature spermatozoa but forward progress in these sperm is very slow. They appear to be continuously bent in one direction, so that their forward movement is erratic. Segment 3 There is not only a sudden increase in the over-all motility of spermatozoa in segment 3 but also a variety of swimming movements. In most preparations, the circular movement seen only occasionally in segment 2 is now the primary pattern. The vibratory motion is still common and in a few preparations it is the dominant type. The circular movement itself shows interesting variations. Some spermatozoa maintain the slow, abrupt form of movement seen in segment 2, while others complete each orbit rapidly and smoothly with a minimum of swimming “strokes.” An occasional sperm may show peculiar darting movement, and a few move forward in a rotatory fashion. The swimming behavior of the rotating sperm is very similar DEVELOPMENT O F SPERM MOTILITY to that of mature sperm except that forward progress is slower and the undulatory waves passing down the flagellum are of greater amplitude. 475 a few show the darting or “tuning f o r k movements. Segment 6 The dominant form of movement in segment 6 is still circular but the width of the orbit shows significant variation. An increased proportion of sperm now display forward movement of either the rotatory or “tuning f o r k type, although forward progress is rarely as rapid as that shown by mature rabbit sperm. There are also a few sperm which show the darting movement. There is considerable variability in the type of swimming movement shown by individual spermatozoa from segment 6. For example, the flagellum of a spermatozoon swimming in tight circles occasionally straightens out, causing the sperm to swim in wide arcs in the “tuning fork” manner. On the other hand, a sperm moving forward in a rotatory manner occasionally stops and begins the circular motion once more. Segment 4 Segment 4 is the principal portion of the caput epididymidis. The tubules are wide and packed with spermatozoa. On release into a fluid medium, nearly all of them show much greater activity than those from preceding segments. They move in a highly localized, circular fashion. In a few spermatozoa, the bend in the flagellum is not so conspicuous and these move in rather wider circles. Only a few sperm show a pattern of movement other than the circular one. An occasional one swims forward with rotation of the head but its progress is rarely as rapid as that of a mature sperm recovered from the ductus deferens. Indeed a sperm such as this sometimes stops moving forward and shows restricted vibratory motion, or begins swimming in a circular fashion. There are a few sperm which show either the “tuning f o r k or darting movement, and an occasional one which Segment 7 More than half of the spermatozoa restill shows the weak, vibratory motion so characteristic of sperm recovered from seg- covered from segment 7 still move in a circular fashion, but the proportion movments 1 and 2. The cytoplasmic droplet is located at the ing in wide circles has increased sigjunction of the midpiece and the mainpiece nificantly. The tendency to move in wide in most sperm. Often it is arranged asym- circles is accompanied by a noticeable inmetrically and the flagellum appears to crease in the rate of flagellar movement. Several spermatozoa now move forward curve towards the side to which the dropwith either the rotatory or the “tuning let is attached. f o r k movement but the proportion showSegment 5 ing forward progress varies considerably Fewer spermatozoa can be recovered between animals. The speed of forward from segment 5 than from segment 4. The movement varies also but it is rare to find primary swimming movement is again cir- sperm moving forward as rapidly as those cular, but the diameter of the circular orbit recovered from the ductus deferens. shows more variation than in sperm from segment 4. While most sperm still move Segment 8 Segment 8 is a transitional zone in which in tight circles, a significant number now swim in rather wider circles. This change a variety of movements may be seen. For is associated with a lesser curvature of the the first time the greater proportion of flagellum. In some preparations, the dif- spermatozoa are now moving forward ference between segments 4 and 5 is re- rather than in a circular fashion. Forward markable in that all the sperm appear to movement of both the rotatory and the “tuning fork” type occurs. Circular swimbe moving in wider circles. Whatever the width of the circle, the ming activity is still present and the width heads of the spermatozoa do not generally of the circular orbit varies considerably. rotate. Only an occasional spermatozoon It is noteworthy that some sperm still swim moves forward with the head rotating and in tight circles. Nearly all preparations 476 PENELOPE GADDUM contain a few sperm showing the darting movement. Segment 9 and ductus deferens The from these show no appreciable difference in their pattern of motility. The majority of them move forward. The circular and darting movements occur rarely and in several preparations they are limited to only a few individuals in each microscopic field. The dominant type of forward movement varies from one animal to another. In many cases the rotatory and the "tuning f o r k types occur in approximately equal numbers, but in some the rotatory type predominates. DISCUSSION As rabbit spermatozoa pass through the male reproductive tract they undergo a remarkable change which enables them to move forward rapidly and efficiently. The relative proportion of sperm showing the typical forward movement increases only gradually as they pass through the epididymis. The achievement of mature swimming behavior in an individual spermatozoon is apparently a slow process. In samples from the caput and corpus epididymidis, for instance, spermatozoa may be seen which are capable of moving forward but only in an abrupt, inefficient manner, with a tendency to revert occasionally to the circular swimming pattern. Even though the change in swimming movements is a gradual one, the over-all pattern of sperm motility in a given segment is sometimes remarkably different from one segment to the next. For example, the spermatozoa from the caput flexure (segment 3 ) show a dramatic increase in activity when compared to those from preceding segments. Sperm from the distal limb of the caput epididymidis (segment 4 ) show an even greater increase in motility. There are relatively few changes in motility pattern in segments 5 , 6 and 7. In segment 8, however, a large proportion of spermatozoa show forward movement. As this study has shown, the maturation of the swimming pattern in rabbit spermatozoa involves a change from a weak, localized, vibratory movement to a swift, circular one and later to a rapid, progres- sively forward movement. A similar observation has been noted briefly by OrgebinCrist ('67b). This sequence of changes is mirrored closely in the maturation of both rat sperm (Blandau and Rumery, '64) and guinea pig sperm (personal observation), despite obvious differences in the mechanism of forward movement shown bv the mature sperm of all these species. This suggests that the essential nature of the change in swimming activity, as described in this paper, may not be confined to rabbit sperm but may be fundamental to the maturation of other mammalian sperm as well. Blandau and Rumery ('64) proceeded to correlate the change in swimming movements of rat sperm with the acquisition of fertilizing capacity. Recently, studies have been completed on the fertilizing capacity of rabbit epididymal sperm as well (Bedford, '66; Orgebin-Crist, '67b). By inseminating spermatozoa removed from various levels of the epididymis into either the oviduct (Bedford, '66) or the cornua (Orgebin-Crist, '67b), these authors have shown independently that fertilizing capacity of rabbit sperm is developed only as they reach the lower half of the corpus epididymidis. The development of fertilizing capacity in the lower corpus epididymidis is remarkable, for the rate of fertilization reported with these sperm was never less than 57%. Our studies did not reveal a noticeable change in the motility pattern in this segment which could account for this sudden rise in fertility. Nevertheless, both reports provided some evidence that significant numbers of the spermatozoa in the lower corpus are still not fully mature. When sperm from this segment were used for oviductal insemination, a 97% fertilization rate was achieved but the ova recovered from these oviducts did not possess as many accessory spermatozoa as those which had been exposed to a similar number of sperm taken from the cauda epididymidis (Bedford, '66). The author suggested that the cauda contains a higher percentage of mature spermatozoa than the lower corpus. Orgebin-Crist ('67b) performed inseminations via the cornua and found that the fertilization rate increased from 57% when spermatozoa from the lower corpus epididymidis were used, to 95% when spermatozoa from the distal DEVELOPMENT O F SPERM MOTILITY cauda epididymidis were inseminated. As seen in this study, the greater proportion of sperm in the lower corpus still swim in a circular fashion, providing additional evidence that full maturity is not attained in this segment. The development of competent forward movement by large numbers of sperm occurs only as they enter the upper coils of the cauda epididymidis. When observed changes in swimming movements are correlated with fertilizing capacity, it becomes obvious that the vibratory and circular movements of sperm from the upper portions of the epididymis are associated with infertility. In contrast, the progressive, rotatory motility found at lower levels of the tract is associated with fertilizing ability. Forward movement may be important for transport through certain portions of the female tract, or for establishing contact with the egg, or both. Blandau and Rumery ('64) showed that rat spermatozoa from the head of the epididymis do not pass through the uterotubal junction. Orgebin-Crist ('67b), on the other hand, believes that rabbit sperm from the caput epididymidis may enter the oviduct readily, suggesting that the uterotubal junction in this animal is less effective as a barrier to sperm transport than it is in the rat. In any event, it seems highly unlikely that the localized, circular movements of immature rabbit spermatozoa would allow frequent collisions with the eggs. Even when such sperm are put in the vicinity of the eggs by intra-tuba1 insemination, they do not effect fertilization (Bedford, '66). Despite the apparent significance of progressive movement for fertilization, this alone is insufficient to ensure full fertilizing capacity. When sperm are trapped by ligation within the first portion of the epididymis (segments 1 and 2, fig. 1) many become capable of rapid, progressive movement in vitro, but they rarely become fertile (Bedford, '67; Orgebin-Crist, '67a). Other factors besides competent motility must be essential in the maturation process. There is some evidence, for instance, that the plasma membrane remains in an immature state in sperm confined within the upper segments of the epididymis, despite the attainment of a progressive motility pattern. This factor may prevent the 477 sperm from establishing contact with ova (Bedford, '67). The circular movements shown by immature rabbit spermatozoa cannot be fully explained as yet, although the motility patterns of mature sperm have been studied in some detail in several species. It has been shown that the swimming motion of mature sperm has two major components, lateral bending and rotation about the longitudinal axis (Bishop, '62). In many species, the primary bending waves are slightly asymmetrical; that is, the amplitude is greater on one side of the flagellum than on the other (Gray, '55; Bishop, '58; van Duijn, van Rosmalen and van Voorst, '66). When a spermatozoon of this type comes close to a glass surface or to the interface between fluid and air, it is prevented from rotating around its longitudinal axis. Then the asymmetry of the bending waves causes the spermatozoon to move along the arc of a circle rather than in a straight line. This may be the explanation for the "tuning f o r k type of movement described above, particularly since the spermatozoa displaying this type of movement are found mainly near the glass surface. The rotational component of flagellar movement normally counteracts the tendency to be propelled in a circle. In the present study, rotation was evident only in those spermatozoa which were moving forward; those that moved in tight circles did not rotate. It may be possible that the two components of sperm movement, lateral bending and longitudinal rotation, are controlled by separate mechanisms which develop at different time intervals during maturation. Perhaps spermatozoa from the head of the epididymis move in tight circles because they are capable of effecting only the lateral bending movements. The rotational component which would correct this asymmetry had not developed. The tendency to swim in circular orbits with no longitudinal rotation is not confined to epididymal spermatozoa. It has been reported as occurring occasionally in spermatozoa from the ductus deferens of guinea pigs (Cody, ' 2 5 ) and in a small proportion of the sperm in human and bovine semen (Farris, '50; Rikmenspoel, 478 PENELOPE GADDUM ’62; Tampion and Gibbons, ’63). Such sperm are generally considered to be pathological, for even sperm which are swimming normally in bovine ejaculates can be induced to swim in circles by subjecting them to cold shock (Rikmenspoel, ’62). Van Duijn (’66) has observed that stiffness in the midpiece of ejaculated spermatozoa leads to circular movement with no rotation, and he believes that this stiffness is often due to some kind of pathological change in the midpiece. The circular movement observed by these workers appears to be similar to that described for spermatozoa from the caput epididymidis in this paper. The possibility that caput spermatozoa are merely “cold shocked is remote since all specimens were prepared at the same temperature. In addition, it has been shown that epididyma1 spermatozoa in a number of species are more resistant to cold shock than ejaculated spermatozoa (Mann, ’64) and that spermatozoa from the caput, in the boar at least, are even more resistant than those from the lower levels of the tract (Lasley and Bogart, ’44). A more likely explanation may be that in ejaculated spermatozoa which have undergone cold shock, the mechanism controlling rotation has been seriously disturbed, whereas in immature spermatozoa from the caput epididymidis, the mechanism simply has not been developed. We believe that the few spermatozoa showing circular movement in the ejaculate may be immature forms which have passed through the entire epididymis without attaining the mature type of swimming behavior. The significance of the changes in motility which occur as sperm pass through the male reproductive tract will not be fully appreciated until further experiments reveal the biochemical or biophysical alterations within the flagellum, or in the environment, which affect the motility pattern. ACKNOWLEDGMENTS The author wishes to thank Dr. Richard J. Blandau for his valuable advice and help in this study and also Mr. Roy Hayashi for his skilled technical assistance in the filming of the sperm movements. LITERATURE CITED Austin. C. R.. and C. S. Sausford 1951 The development ‘of the rat spermatid. J. Roy. Micr. SOC.,71: 397406. Bedford, J. M. 1965 Changes i n fine structure of the rabbit sperm head during passage through the epididymis. J. Anat., 99: 891-906. -1966 Development of the fertilizing ability of spermatozoa in the epididymis of the rabbit. J. Exp. Zool., 163: 319-329. 1967 Effects of duct ligation on the fertilizing ability of spermatozoa from different regions of the rabbit epididymis. J. Exp. Zool., 166: 271-282. Bishop, D. W. 1958 Motility of the sperm flagellum. Nature (London), 182: 1638-1640. 1961 Biology of spermatozoa. In: Sex and Internal Secretions, 3rd edition. W. C. Young, ed. Williams and Wilkins, Baltimore, Volume 2, pp. 709-710. 1962 Sperm motility. Physiol. Rev., 42: 1-59. Bishop, M. W. H., and A. Walton 1960 Spermatogenesis and the structure of mammalian spermatozoa. In: Marshall’s Physiology of Reproduction, 3rd edition. A. S. Parkes, ed. Longmans Green, London, Volume 1, part 2, pp. 95-96. Blandau, R. J., and R. E. Rumery 1964 The relationship of swimming movements of epididyma1 spermatozoa to their fertilizing capacity. Fertil. Steril., 15: 571-579. Cody, B. A. 1925 Observations and experiments upon spermatozoa of the guinea pig. J. Urol., 13: 175-191. Duijn, C. van, Jr. 1966 Normal and abnormal patterns of spermatozoan movement in relation to longevity and fertilizing power. Acta Physiol. Pharmacol. Neerl., 13: 478480. Duijn, C. van, Jr., W. van Rosmalen and C. van Voorst 1966 Cinematographical model studies of normal and abnormal movements of spermatozoa. Res. Film, 5: 622-641. Farris, E. J. 1950 Human fertility and problems of the male. The Author’s Press, Inc., White Plains, New York, pp. 58-61. Fawcett, D. W., and R. D. Hollenberg 1963 Changes in the acrosome of guinea pig spermatozoa during passage through the epididymis. Z. Zellforsch., 60: 276-292. Gaddum, P. 1967 Changes in the swimming movements of rabbit spermatozoa during maturation. Anat. Rec., 157: 359 (Abstract). Gray, J. 1955 The movement of sea-urchin spermatozoa. J. Exp. Biol., 32: 775-801. Lasley, J. F., and Bogart, R. 1944 Some factors affecting the resistance of ejaculated and epididymal spermatozoa of the boar to different environmental conditions. Amer. J. Physiol., 141: 619-624. Mann, T. 1964 The biochemistry of semen and of the male reproductive tract. Methuen, London. Wiley and Sons, New York, p. 54. Nicander, L. 1957 On the regional histology and cytochemistry of the ductus epididymidis in rabbits. Acta Morph. Neerl. Scand., 1: 99118. DEVELOPMENT O F SPERM MOTILITY Orgebin-Crist, M. C. 1967a Sperm maturation in rabbit epididymis. Nature (London), 216: 816-818. 1967b Maturation of spermatozoa in and the rabbit epididymis: fertilizing embryonic mortality in does inseminated with epididymal SPematoz'Ja. Ann. Bid. h i m . Biochim. Biophys., 7: 373-389. Rikmenspoel, R. 1962 Biophysical approaches to the measurement of sperm motility. In: 479 Spermatozoan Motility. D. W. Bishop, ed. Amer. ASS. Adv. Sci., Washington, D. C., no, 72, pp. 31-54. Tampion, D., and R. A. Gibbons 1963 Effect of pH on the swimming rate of bull spermatozoa. J' Reprod' Ferti1'9 5: 249-258' Young, W. C. 1931 A study of the function of the epididymis. 111. ~ ~ ~changes ~ undert i gone by spermatozoa during their passage through the epididymis and vas deferens in the guinea pig. J. Exp. Biol., 8: 151-162. ~ PLATE 1 EXPLANATION OF FIGURES 2-5 480 Sequences in the circular movement of a spermatozoon recovered from the caput epididymidis. (Original magnification X 1060). DEVELOPMENT O F SPERM MOTILITY Penelope Gaddum PLATE 1 481 PLATE 2 DEVELOPMENT OF SPERM MOTILITY Penelope Gaddum EXPLANATION O F FIGURE 6 482 A spermatozoon recovered from the ductus deferens which was showing normal, progressive rotatory movement. (Original magnification X 1060).