epididymis, respectively. The occurrence and disposition of the droplet

Transcription

1 MORPHOLOGICAL CHANGES IN RABBIT SPERMATOZOA DURING PASSAGE THROUGH THE EPIDIDYMIS J. M. BEDFORD Department of Physiology, Royal Veterinary College, London, N. W. 1 {Received 28th August 1962) Summary. The fertilizing ability of rabbit spermatozoa is acquired in passage through the epididymis, during which certain changes take place in the spermatozoon. It has been found here that a significant diminu tion ( <0 01) in acrosome width and length occurs during passage of spermatozoa from caput to cauda epididymis. Within-group analysis of the caput sperm group reveals a significant correlation ( <0 01) of acrosome width with the position of the cytoplasmic droplet on the midpiece. A large difference in the cytoplasmic droplet distribution is apparent between samples of spermatozoa from the cauda and caput epididymis, respectively. The occurrence and disposition of the droplet in either of these regions was not obviously altered following a series of successive ejaculations. The change in acrosome size during epididymal passage is discussed in relation to the development of fertilizing ability of rabbit spermatozoa. INTRODUCTION It is well recognized that during passage from the caput to the cauda epididy mis, mammalian spermatozoa undergo a 'ripening' process with respect to their fertilizing ability. Young (1931) showed that when spermatozoa from the proximal part of the guinea-pig epididymis were inseminated into oestrous females, 33 % became pregnant as compared with 68 % when spermatozoa from the cauda epididymis were used for insemination. In the rabbit, it has been found (unpublished results) that following vaginal insemination or instillation of caput spermatozoa directly into the Fallopian tubes, 7 to 8 hr prior to ovulation, only 3-6% of 142 ova were fertilized. On the other hand, the use of a similar concentration of cauda spermatozoa in this way resulted in 98 % of fifty-one eggs being fertilized. It seems probable that the acquisition of fertilizing ability by spermatozoa, during passage from the caput to the cauda epididymis, results from the maturation of a number of the systems which together ensure the functional integrity of the spermatozoa. As yet, however, only a few of the distinguishing characteristics between caput and cauda spermatozoa have been recognized. Included in these are motility (Redenz, 1926) and resistance to heat (Young, 1929) and to cold shock (Lasley & Bogart, 1944). It has also been suggested ß* 169

2 170 J. M. Bedford that bull spermatozoa show an increase in specific gravity with maturation (Lindahl & Kihlstrom, 1952). Morphologically it appears that, in human spermatozoa at least, changes occur in the structural organization of the mitochondria in the midpiece during passage of the spermatozoon through the epididymis (Anberg, 1957; D. W. Fawcett cited by Bishop, 1961). Another structural feature, the cytoplasmic droplet, has received attention from various authors. The droplet was described by Retzius (1909), and later Redenz (1926) and others noted that the droplet moves down the midpiece from the base of the head and is lost from most spermatozoa during passage through the epididymis. Hancock (1955) has shown that in the bull the migration of the droplet occurs almost exclusively within the caput epididy mis. The role of the droplet, if any, remains obscure. Up to the present time, the change in position and subsequent loss of the droplet is the only gross morphological change which has been observed in the mammalian spermatozoon during its epididymal passage. This communication describes a significant difference in acrosome size, which is correlated in part with the position of the cytoplasmic droplet, and which has been found between spermatozoa from different regions of the epididymis. The effect of rapid successive ejaculations upon the occurrence and location of the cytoplasmic droplet in spermatozoa from these regions has also been studied. MATERIALS AND METHODS In these investigations, adult male rabbits of different breeds were used. For each series of observations one epididymis was removed from a live animal under pentobarbitone anaesthesia, and immediately immersed in Ringer's solution at 25 C. The caput and cauda regions of the epididymis were then severed at the levels indicated in PL 1, Fig. 1. To release the spermatozoa these tissues were cut with scissors into a number of pieces, and were pressed, forcing the spermatozoa from the ducts into the medium. The suspension of sperma tozoa so produced was then adjusted to a concentration of approximately 1 million spermatozoa/ml. For examination of the spermatozoa, thin smears were fixed on glass slides by drying in air, and stained in Rose Bengal (1 %) for 3 to 4 hr. The slides were then removed from the stain and were stacked vertically and dried in air for 3 to 4 hr. For measurement and photography, spermatozoa were viewed under oil at a magnification of X 1200 in bright field, with a light source provided by a mercury arc vapour lamp. Photographs were taken with Kodak P.300 rapid panchromatic plates. In each of four animals, measurements were made of the length and width of the acrosome (Text-fig. 1) in random samples of spermatozoa taken from the caput and from the cauda epididymis, respectively. The head length also was in the cauda and in some of the caput spermatozoa from each animal. However, the presence of the droplet at the neck precluded measurement of head length in many caput spermatozoa. The measurements were made in a darkened room by an arrangement (Text-fig. 2) in which the image (S) of the spermatozoon was reflected through a prism eye piece attachment (P) onto a surface mirror (M) and thence onto

3 PLATE 1 Fig. 1. Rabbit testis and epididymis showing the levels from which caput spermatozoa (A) and cauda spermatozoa (B) were taken. Fig. 2. Spermatozoa from caput epididymis showing swollen acrosome and cytoplasmic droplet at base of head. Rose Bengal, Fig. 3. Spermatozoa from cauda epididymis with retained midpiece droplet. Rose Bengal, (Facing p. 170)

4 Changes in rabbit spermatozoa in the epididymis 171 standard millimetre graph paper (I). The distances between these points were constant throughout the series of measurements. The spermatozoa were viewed under oil immersion, and on each slide two longitudinal axes only were traversed. By avoidance of the initial oil mark no one spermatozoon was included twice. Before measurement the outline of each spermatozoon was Text-fig. 1. Diagrammatic representation of rabbit sperm head showing the para meters of acrosome length (L), width (W) and head length (H). M Text-fig. 2. Diagram of the sperm-image magnifying system. S slide image; prism = = attachment; M surface reflection mirror; I final image projected = onto graph = paper. focused sharply and. the length and width of the magnified acrosome image was then recorded on the graph paper. From measurements with a micrometer slide, it was established that a distance of 1 mm on the graph paper at the final image point was equivalent to 0-46 µ at the surface of the microscope slide. Employing this constant, absolute values for the sperm-head dimensions were calculated; these are shown in Tables 1 to 3. The significance of the difference in the means of the

5 172 J. M Bedford sperm-head parameters was obtained from an analysis of variance. It is recognized that biological measurements of this type may lend themselves to unconscious bias on the part of the observer. Unfortunately, 'blind' measure ments were not possible as the presence and position of the cytoplasmic droplet served easily to identify the particular population being assessed. In studying the effect of ejaculation upon droplet distribution in different regions of the epididymis, a procedure similar to that described previously was used to obtain and to prepare the spermatozoa, except that the left epi didymis was removed within 15 min of six rapid successive ejaculations achieved with an artificial vagina (Macirone & Walton, 1938). After 3 weeks sexual rest, samples of spermatozoa were then prepared from the contralateral epididymis of each animal. For estimation of the droplet distribution in any sperm population, the spermatozoa were observed directly at a magnification of 840, with an orange (OY.2) filter. Two hundred and fifty spermatozoa were observed in each group. The droplet distribution was classified according to the position of the cyto plasmic droplet. The classes recorded were: spermatozoa having the droplet at the base of the head or neck of the midpiece ('head' droplet spermatozoa) ; spermatozoa in which the droplet was situated at some point along the tail midpiece ('midpiece' droplet spermatozoa) ; and spermatozoa in which the droplet was absent ('no droplet' spermatozoa). RESULTS The mean values for width and length of the acrosome in spermatozoa samples from the caput and cauda epididymis are shown in Table 1. In each male the Table 1 comparison of acrosome dimensions in spermatozoa from caput and cauda epididymis Parameter Buck No. Caput spermatozoa Spermatozoa Mean±s.E. Cauda spermatozoa Spermatozoa Mean ±s.e. (t>) Difference between means Significance difference Acrosome width 5-24 ± ± ± ± ± ± ± ± Acrosome length 4-52 ± ± ± ± ± ± ± ± <0 01 /><0-01 means of these dimensions in caput spermatozoa were significantly greater () than those in spermatozoa from the cauda region of the same epididymis. In Table 2 within-group analysis of the caput spermatozoa, in relation to the position of the cytoplasmic droplet, revealed a significantly greater mean acrosome width () in those spermatozoa having the

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7 174 J. M. Bedford droplet at the base of the head than in those in which the droplet at some point along the midpiece. length between these groups was significant in only was situated The difference in the means of acrosome two of the four animals. Table 3 comparison of acrosome dimensions and head length in 'midpiece' and NO droplet' caput spermatozoa, with those in cauda spermatozoa Parameter Buck No. 'Midpiece' and 'no droplet' caput spermatozoa Spermatozoa Mean ±s.e. ( 0 Cauda spermatozoa Spermatozoa Mean±s.K. (µ) Difference in means (µ) Significance of difference Acrosome width ± ± ± ± ± ± ± ± Acrosome length ± ± ± ± ± ± ± ± P<010 /><001 Head length ± ± ± ± ± ± ± ± Table 4 distribution of cytoplasmic droplet in spermatozoa from caput and cauda epididymis respectively Buck No. Spermatozoa sample A Head Caput epididymis Midpiece No droplet Head 14-0 Cauda epididymis Midpiece No droplet A A A Mean A Two hundred and fifty spermatozoa counted in each group. A = Spermatozoa collected after 3 weeks sexual rest. = Spermatozoa collected 15 min after six successive ejaculations. With the exception of Buck 2, no significant difference was apparent in the means of acrosome width or length of the 'midpiece' and 'no droplet' groups. Nevertheless, from Table 3, it is clear that considering only the 'midpiece' and

8 Changes in rabbit spermatozoa in the epididymis 175 'no droplet' caput spermatozoa, the means of acrosome width and length in these were still significantly greater than the corresponding means for sperma tozoa from the cauda region of the same epididymis. In Table 3, the values for mean head length of cauda spermatozoa, which are similar to those for ejaculate spermatozoa reported by Beatty & Napier (1960), are significantly less than the values for the corresponding caput spermatozoa. At present it is not possible to say whether the greater mean head length of the latter group is, in part, an absolute difference, or whether this is merely a reflection of greater acrosome thickness over the anterior surface of the head of the spermatozoon. The data in Table 4 show that there were marked differences in the droplet distribution between spermatozoa samples from the caput and cauda epididy mis. However, the occurrence and position of the droplet in spermatozoa from the caput epididymis, taken within 15 min of six rapid successive ejaculations in each of four males, was not markedly different to that in spermatozoa from the caput of the contralateral epididymis taken after 3 weeks sexual rest. Moreover, with the possible exception of Animal 7, no difference was seen in the droplet distribution in spermatozoa samples from the cauda epididymis of these males. DISCUSSION The data in Table 1 show clearly that in each buck the mean acrosome size in a sample of caput spermatozoa was significantly greater than that of sperma tozoa taken from the cauda region of the same epididymis. This difference can be seen in a comparison of the sperm heads in PL 1, Figs. 2 and 3. A breakdown analysis of the caput-sperm groups, with respect to the position of the droplet, has revealed that the width of the acrosome of the 'head droplet' spermatozoa was significantly greater than that of either the 'midpiece' or 'no droplet' spermatozoa. This would seem to support the commonly held view that the presence and location of the cytoplasmic droplet may be taken as an approxi mate indication of the relative maturity of epididymal spermatozoa. No significant difference in mean acrosome size was apparent between the caput 'midpiece' and 'no droplet' spermatozoa in three of the four animals studied. Nevertheless, the mean acrosome size of the combined caput 'midpiece' and 'no droplet' spermatozoa groups remained significantly greater than the acrosome size of the cauda spermatozoa (Table 3). It would thus appear that at about the time the droplet leaves the head, a marked reduction in acrosome size occurs and that this continues as a more gradual process as the cauda is approached, where finally the majority of spermatozoa achieve functional maturity. It has been shown that during spermateliosis in man, monkey and ram (Clermont & Leblond, 1955) and in the cat (Burgos & Fawcett, 1955), the acrosomic granule within a vesicle flattens over the anterior nuclear surface of the spermatozoon head, eventually giving rise to the acrosome. Considering the subsequent changes in acrosome dimensions in the epididymis demon strated here, it seems reasonable to suppose that in the rabbit, at least, the acrosome does not reach a state of functional maturity in the testis, and that

9 176 J. M. Bedford this is achieved only during epididymal passage as the acrosome contracts, becoming apparently more closely apposed to the nuclear surface of the sperm head. The acrosome has received much attention and various functions have been ascribed to it. Cases of sterility in bulls (Teunissen, 1946; Hancock, 1949) and boars (Bane, 1961) have been shown to be associated with hereditary acro some defects. It has been suggested also (Austin & Bishop, 1958) that changes occur in the acrosome during capacitation, a necessary preliminary for fertili zation in the rabbit (Austin, 1951; Chang, 1951). In the light of these con siderations, it seems reasonable therefore to suppose also that reduction in acrosome size during epididymal passage is an essential preliminary to the acquisition of fertilizing ability by rabbit epididymal spermatozoa. If it is assumed that sperm maturation follows a similar course in the epididy mis of the bull and the rabbit, the decrease in size of the acrosome, and perhaps in head length, of the rabbit spermatozoa during this epididymal transit, might add support to the claim of Lindahl & Kihlstrom (1952), that the specific gravity of bull spermatozoa increases during the maturation process in the epididymis. Lindahl & Kihlstrom (1952) suggested that the increase in specific gravity of epididymal spermatozoa in the bull is associated with a process of dehydration. Whether the reduction in size of the rabbit acrosome during epididymal passage is due to dehydration remains to be determined however. A marked difference in the distribution of the was cytoplasmic droplet evident between the populations of spermatozoa from the caput and cauda regions of the epididymis, respectively. However, the fact that, with the possible exception of one animal, no significant change was observed in the droplet distribution following a number of successive ejaculations suggests that sperm passage in the upper reaches of the epididymis is not markedly influenced by ejaculation over any short period. The droplet distribution in the caput region of the rabbit epididymis (Table 4) shows a close similarity to published figures for the bull epididymis (Branton & Salisbury, 1947), but the midpiece droplet appears to be retained in a greater proportion of spermatozoa in the cauda epididymis of the bull. ACKNOWLEDGMENTS I would like to thank Professor E. C. Amoroso, f.r.s. for the provision of research facilities in his department and for criticism of the manuscript. I would also like to thank Dr M. W. H. Bishop and Mr C. A. Finn for helpful discussion. REFERENCES Anberg, A. (1957) The ultrastructure of the human spermatozoon. An electron microscope study of spermatozoa from sperm samples and the epididymis, including some observations of the spermatid. Acta obstet, gynec. scand. 36, Suppl. 2. Austin, C. R. (1951) Observations on the penetration of the sperm into the mammalian egg. Aust. J.sci. Res. B, 4, 581. Austin, C. R. & Bishop, M. W. H. (1958) Role of the rodent acrosome and perforatorium in fertiliza tion. Proc. roy. Soc., 149, 241. Bane, A. (1961) Acrosome abnormality associated with sterility in the boar. Proc. IVth int. Congr. Anim. Reprod., The Hague, p. 810.

10 Changes in rabbit spermatozoa in the epididymis 177 Beatty, R. A. & Napier, R. A. N. (1960) Genetics of gametes. II. Strain differences in characteristics of rabbit spermatozoa. Proc. roy. Soc. Edinb. B, 68, 17. Bishop, D. W. (1961) Biology of spermatozoa. Sex and Internal Secretions, 3rd edn., vol. 2, p Ed. W. C. Young. Baillière, Tindall & Cox, London. Branton, C. & Salisbury, G. W. (1947) Morphology of spermatozoa from different levels of the reproductive tract of the bull. J. Anim. Sci. 6, 154. Burgos, M. H. & Fawcett, D. W. (1955) Studies on the fine structures of the mammalian testis. I: Differentiation of spermatids in the cat (Felis domestica). J. biophys. biochem. Cytol. 1, 287. Chang, M. C. (1951) Fertilizing capacity of spermatozoa deposited into the Fallopian tubes. Nature. Lond. 168, 697. Clermont, Y. & Leblond, C. P. (1955) Spermiogenesis of man, monkey, ram and other mammals as shown by the 'Periodic acid Schiff'technique. Amer. J. Anat. 96, 229. Hancock, J. L. (1949) Evidence of an inherited seminal character associated with infertility of Friesian bulls. Vet. Ree Hancock, J. L. (1955) The disintegration of bull spermatozoa. Vet. Ree. 67, 825. Lasley, J. F. & Bogart. R. A. (1944) Some factors affecting the resistance of ejaculated and epididymal spermatozoa of the boar to different environmental conditions. Amer. J. Physiol. 141, 619. Lindahl, P. R. & Kihlstrom, J. E. (1952) Alterations in specific gravity during the ripening of bull spermatozoa. J. Dairy Sci. 35, 393. Macirone, C. & Walton, A. (1938) Fecundity of male rabbits as determined by 'dummy' matings. J. agrie. Sci. 28, 122. Redenz, E. (1926) Nebenhoden und Spermienbewegung. Wurzb. Abhundl. gesamt. Med. n.f., 4, 107. Retzius, G. (1909) Die Spermien der Nagetiere. Biol. Untersuch. N.F.Jena, 14, 133. Teunissen, G.. B. (1946) Een afkijking van het acrosoom (kopkap) bij de spermatozoiden van een stier. Tijdschr. Diergeneesk. 71, 292. Young, W. C. (1929) The influence of high temperature on the reproductive capacity of guinea-pig spermatozoa as determined by artificial insemination. Physiol. ool. 2, 1. Young, W. C. (1931) A study of the function of the epididymis. III. Functional changes undergone by spermatozoa during their passage through the epididymis and vas deferens in the guinea-pig. J. exp. Biol. 8, 151.