A Study of the Function of the Epididymis
1931; The Company of Biologists; Volume: 8; Issue: 2 Linguagem: Inglês
10.1242/jeb.8.2.151
ISSN1477-9145
Autores Tópico(s)Reproductive Biology and Fertility
ResumoIn two recent papers (Young, 1929 a, b) attention was directed to the fact that, in mammals, spermatozoa which appear to have attained their full structural development while still attached to the germinal epithelium, are compelled to pass through the long coiled ductus epididymidis before they reach the vas deferens where they are in a position to be discharged. It was noted further that, while the significance of the epididymis for spermatozoa and the nature of changes undergone by spermatozoa during their passage through this organ have been subjects of experimentation by numerous investigators, current opinions with respect to these questions are both numerous and varied.Among other opinions, the suggestions had been made (1) that certain developmental changes which are important for the successful functioning of spermatozoa are attributable to some specific action of the epididymal secretion (Tournade, 1913; Stigler, 1918; Braus and Redenz, 1924; Redenz, 1924, 1925 a, b, 1926; von Lanz, 1924 b, 1926), and (2) that the epididymis is a reservoir functioning to preserve the vitality of spermatozoa stored in it until the time of their discharge (Van der Stricht, 1893; Tournade and Delacarte, 1913; Braus and Redenz, 1924; Redenz, 1924, 1925 a, b, 1926; von Lanz, 1924 a, b, 1926). These opinions have since been reaffirmed and extended (von Lanz and Malyoth, 1928; von Lanz, 1929; Redenz, 1929; Redenz and Belonoschkin, 1929; and Belonoschkin, 1929 a, b).What appeared to be a defect in the first of these theories was the fact that certain developmental changes, such as the acquisition of the capacity for being stimulated to motion and, to some extent, the increased capacity for resisting high temperature, which are undergone by spermatozoa during their passage through the epididymis, represent nothing more than a continuation of changes which are occurring while the spermatozoa are still contained in the testis (Young, 1929 a). Such being the case, it seemed unnecessary to postulate the existence of some stimulus to spermatozoon development in the epididymis which is not present in the testis. The second theory, to the effect that the epididymis functions to preserve the vitality of spermatozoa until the time of their discharge, was questioned after it had been found that any preserving action possessed by the epididymal secretion is powerless to prevent the ageing of spermatozoa which normally seems to follow the attainment of their maturity (Young, 1929 b).Because of these objections to the older conceptions of the epididymal spermatozoon relationship, a new theory was formulated as far as this was possible on the basis of the experiments which had been performed. It was suggested that the epididymis provides an environment in which stimuli to spermatozoon development which are not different from those found in the testis are present; that spermatozoa require more time for the completion of their development than they have before they are loosened from the germinal epithelium; that they are, therefore, carried into the epididymis as immature or unripe cells which are incapable of functioning; and that much of the time consumed by them in passing through the epididymis is necessary for the completion of their development. Once functional maturity has been attained, however, there is no influence which preserves them indefinitely and, unless they are discharged, they age and become at first incapable of effecting fertilisation and finally incapable of being stimulated to motion. In other words, it was suggested (1) that the developmental changes which occur in spermatozoa during their passage through the epididymis are not conditioned by some specific action of the epididymal secretion, but are inherent in the protoplasm of the spermatozoa themselves, and (2) that emphasis should be shifted from the conception of the epididymis as a spermatozoon reservoir acting to preserve spermatozoa which have attained a certain level of development in a state of static maturity to the conception of the epididymis as an organ of spermatozoon development in which these cells are constantly changing; ripening until an optimal functional capacity is attained and then ageing if their residence is prolonged.As indicated in a brief, preliminary communication (Young and Simeone, 1930), confirmation and extension of these suggestions have come from two groups of experiments. The first involved a study of the reproductive capacity of spermatozoa removed from two different levels of the epididymis of the guinea-pig under normal and experimental conditions. A description of the methods which were employed along with an enumeration of the results which were obtained, and a discussion of the application of these results to the problem as a whole, form the contents of this paper.The second group of experiments involved an investigation of the fate of non-ejaculated spermatozoa. The observations made during this part of the study are reported elsewhere (Simeone and Young, 1931).The changes which occur in the reproductive capacity of spermatozoa as they pass through the epididymis were determined by means of the artificial insemination of females with spermatozoa from two levels of the epididymis under normal and experimental conditions. If it is true, as has been supposed, that spermatozoa enter the epididymis as immature cells which ripen and acquire their capacity for effecting fertilisation as they pass through this organ, the percentage of fertile inseminations when younger spermatozoa from the proximal end of the epididymis are used should be less than the percentage of fertile inseminations when older spermatozoa from the distal end are used.The procedure which was employed in testing this hypothesis was as follows: Whenever two or more females came into heat simultaneously the epididymides from a normal male were separated along the line a…b, Fig. 1. Each part was then macerated in a quantity of Locke's solution so selected that the densities of the resulting spermatozoon suspensions would be as nearly equal as possible. A light ether anaesthesia was then administered to the females to be inseminated, and approximately 12 c.c. of the suspension introduced into the body of the uterus by means of a glass catheter. In this way one female was inseminated with spermatozoa from the proximal end of the cauda epididymidis, and the other with spermatozoa from the distal end of the cauda epididymidis from the same male. The female were then held ventral side up for 5 minutes, after which they were numbered and placed in cages reserved for experimental animals. From the fifteenth to the twentieth days inclusive after the insemination, each female was examined twice daily for rupture of the vaginal closure membrane or other signs of a recurrent oestrum. If such had not occurred by the twentieth day, the female was regarded as pregnant and kept isolated until the end of the gestation period, at which time the size and condition of the litter were noted. Any irregularities such as still-births, abortions, and intra-uterine resorptions were also recorded.At the time of insemination samples of the spermatozoon suspensions were examined microscopically. It is of interest, in connection with a point to be mentioned later that, without exception, the spermatozoa removed from the distal end exhibited a more vigorous motion than those removed from the proximal end.Ninety-nine females were inseminated with spermatozoa removed from the proximal portion of the cauda epididymidis, and ninety-seven were inseminated with spermatozoa removed from the distal portion. Of the former, thirty-three, or 33·3 Per cent., were impregnated. Of the females inseminated with spermatozoa from the distal end, sixty-six, or 68 per cent., were impregnated. The doubling of the number of fertile inseminations, when spermatozoa from the distal portion of the epididymis were used, may be attributable either to the greater maturity of spermatozoa from the distal end or to the maturity of a much larger number of spermatozoa from this portion of the epididymis. In either case the difference is believed to provide ample evidence for the developmental nature of changes which occur in spermatozoa during their passage through the epididymis. The opinion that these changes are inherent in the spermatozoa rather than conditioned by some specific action of the epididymal secretion has been expressed. Additional evidence will be cited elsewhere in the paper.The suggestion (Young, 1929 b) that spermatozoa within the epididymis which have attained an optimal functional maturity are not preserved indefinitely, but soon begin to undergo regressive or degenerative changes, was tested by a modification of the above described procedure.Following an abdominal incision and the retraction of the testis and epididymis into the body cavity, it was possible to ligature the head of the epididymis in one place, c, and the vas deferens in two places, d and e, without interfering with the vascularisation of the structures involved. The testes were then replaced in the scrotal sacs. Provided no adhesions developed, the males were killed 20 and 25 days later, and spermatozoa removed from the proximal and distal ends of the cauda epididymidis as before. At this time samples of the spermatozoon suspensions were examined microscopically.It was found that the condition of the tubules 20 or 25 days after the operation is never similar to that at the time of the operation, provided the ligature of the head of the epididymis has been complete. Most of the tubule proximal to the line a...b has been emptied, and the portion of the tubule distal to the line a...b has become conspicuously distended with spermatozoa which have been forced into it from the proximal levels. Despite this condition it is easily possible to remove spermatozoa from the proximal and distal portions of the column without the contamination of either.It was expected, if the course of changes undergone by spermatozoa during their residence in the epididymis is similar to that which had been postulated, that the younger spermatozoa contained in the proximal end of the cauda epididymidis at the time of the operation would mature, develop the capacity for a more vigorous motion and become more capable of effecting fertilisation. Similarly, it was expected that the older, mature or ripe spermatozoa contained in the distal end of the cauda epididymidis at the time of the operation would age, become weakened in their capacity for being stimulated to motion, and become less capable of effecting fertilisation. In short, it was expected that the conditions with respect to the strength of motility and the capacity for effecting fertilisation would be reversed from what they had been when spermatozoa from normal males were used.Forty-three females were inseminated with spermatozoa from the proximal end of the spermatozoon column, and forty were inseminated with spermatozoa from the distal end of the column. Of the former, nineteen, or 44·2 per cent., were impregnated. Of the females inseminated with spermatozoa from the distal end of the column, thirteen, or 32·5 per cent., were impregnated. In most cases, spermatozoa removed from the proximal end of the column were as active or conspicuously more active than those from the distal end of the column. As was expected, therefore, spermatozoa which have been isolated in the epididymis for 20 days show a reversal of the conditions which exist in the normal epididymis. Spermatozoa at the proximal end of the column can be stimulated to a more vigorous motion, and are more successful in effecting fertilisation after 20 days' isolation in the epididymis than those from the distal end of the column in the same animals.This reversal of the normal relationship was even more striking in the case of spermatozoa which had been isolated 25 days. In this part of the experiment ninety-eight females were inseminated with spermatozoa from the proximal end of the column, and one hundred females were inseminated with spermatozoa from the distal end of the column. Of the former, forty-eight, or 49 per cent., were impregnated. Of the females inseminated with spermatozoa from the distal end of the column, twenty-five, or 25 per cent., were impregnated.The data described above are summarised in the two upper lines of Table I. Based upon actual reproductive capacity, which is unexcelled as a measure of general fitness, they reveal more clearly than any data obtained heretofore the course of changes which can occur in spermatozoa during their residence in the epididymis. Developmental changes culminating in the attainment of an optimal condition for effecting fertilisation are the first to occur. They are followed by regressive changes which result in a loss of the capacity for effecting fertilisation. Proof that developmental changes occur came from the fact that the younger spermatozoa removed from the proximal end of the epididymis were successful in effecting fertilisation in but 33-3 percent, of the cases, while older spermatozoa removed from the distal end of epididymides from the same males were successful in effecting fertilisation in 68 per cent, of the cases. Further proof came from the fact that the ability of the younger spermatozoa to effect fertilisation increased from an approximate normal of 33·3 per cent., under the conditions of the experiment, to 44·2 per cent, at the end of 20 days' isolation in the epididymis and to 49 per cent, at the end of 25 days' isolation. Proof that regressive changes set in following the attainment of an optimal functional capacity came from the fact that the ability of the mature spermatozoa located in the distal end of the epididymis to effect fertilisation decreased from an approximate normal of 68 per cent., under the conditions of the experiment, to 32·5 per cent, following 20 days' isolation in the epididymis and to 25 per cent, following 25 days' isolation.Whether or not the fertilising capacity of spermatozoa from the proximal end of the column would have increased further, and that of spermatozoa from the distal end of the column would have decreased further with 30 days' isolation is not known. It is planned to determine this in an extension of the investigation.That the reversal of the spermatozoon relationships referred to above was not more complete at the end of 25 days may be attributable to certain factors which could not be controlled, namely, a constant interchanging and shifting in position of spermatozoa within the epididymis. Whether or not there is any active motion of spermatozoa within the epididymis which might be partly responsible for this is not known, although Redenz and von Lanz claim that spermatozoa within the epididymis are capable of independent motion. It may be instead that younger spermatozoa are constantly being forced into the distal part of the epididymis and vas deferens to mix with the older spermatozoa by the distally directed muscular pressure. In addition, the removal of old spermatozoa in the epididymis and vas deferens by liquefaction, which is now known to occur there (Simeone and Young, 1931), may leave places which are filled by viable spermatozoa from the proximal regions of the tubule.An observation of interest, in connection with the suggestion that the development of spermatozoa which takes place during their passage through the epididymis is inherent in their protoplasm rather than conditioned by simuli originating in the epididymis, was made on the testes removed from certain males 20–25 days after the ligature of the head of the epididymis. Many such testes contained numerous patches of white seminiferous tubules which bore a striking resemblance to the small portion of the tubule of the cauda epididymidis proximal to the line a...b in the figure. When these white tubules were removed, macerated in Locke's solution, and studied microscopically, they were found to contain many active spermatozoa. This condition was in marked contrast to that found in the seminiferous tubules from a normal testis, where only an occasional spermatozoon can be stimulated to exhibit the weakest flagellation of its tail. It is, however, similar to that found in the testis of the albino rat (Young, 1929 a), where many spermatozoa capable of being stimulated to motion can be found.It is suggested that the great increase in the number of such spermatozoa which can be stimulated to motion and the great increase in the strength of their motility are attributable to the greater maturity of these cells which have been unable to pass from the testis into the epididymis. Presumably, their development has continued despite their retention in the testis. The observation that this continuation of development is not dependent upon the passage of the spermatozoa into the epididymis provides further evidence against the idea of a specific action of the epididymal secretion on spermatozoon development. The extent to which spermatozoa isolated in the testis in this manner can develop is not known.The data (Table I) which have to do with litter size and condition are meagre, since only 204 of the 477 inseminated females were impregnated. They are presented, however, for what they suggest in the way of future experiments rather than for any conclusions which may be drawn from them at this time.Average litter size was smallest, 1·84, among that group of females inseminated with the youngest spermatozoa, namely, those from the proximal end of the cauda epididymidis from normal males. It rose to 2·60, when older spermatozoa from the distal end of the cauda epididymidis were used. It remained near this level, 2·50 and 2·58, when spermatozoa which had been isolated 20 days were used, and decreased slightly to 2·34 and 2·36 when spermatozoa which had been isolated 25 days were used. In experiments which are now in progress an attempt is being made to determine if the smaller litters, which seem to follow inseminations with the youngest and oldest spermatozoa, are attributable to the fertilisation of a smaller number of ova or to an unusually large number of intra-uterine resorptions.The number of still-born individuals on the one hand, and the number of individuals aborted or resorbed within the uterus on the other hand, were recorded separately. The percentage of still-born individuals will be noted to be fairly constant throughout, and as high among the females inseminated with the youngest spermatozoa as among those inseminated with the oldest spermatozoa. The percentage of individuals which were aborted or resorbed, on the other hand, waá1 somewhat lower, 3-6 and 9 per cent., among the controls than it was, 18·8 to 22·7 per cent., when the older spermatozoa were used. It is of interest to note that many of these individuals showed gross structural defects.The question arises: is this larger number of abortious cases of intra-uterine resorption and foetal abnormalities which occurred following the insemination of females with the older spermatozoa due to the age of the spermatozoa which were used? Imperfect development has been suggested as a consequence of the fertilisation of the eggs of certain invertebrates by old spermatozoa (Dungay, 1913, for Nereis; Medes, 1917, for Arbacia), but whether this also applies to the guinea-pig can be determined only after more data have accumulated from experiments now in progress.The uniform frequency with which still-born individuals were born to females in all groups compared with the variation in the frequency with which abortions and intra-uterine resorptions occurred, suggests that these gestational abnormalities may be two different phenomena traceable to different causes rather than being expressions of the same phenomenon differing only in the time of occurrence. The still-births may be a consequence of parturitional difficulties associated, possibly, with the size of the foetus or its position within the uterus. Abortions and intra-uterine resorptions may have, on the other hand, a germinal origin. Again, it is expected that experiments which are now being undertaken, will dispose of this problem one way or another.The data which have been obtained from the experiments described above and from earlier experiments (Young, 1929 a, b) furnish a basis for an evaluation of the divergent theories of the epididymal spermatozoon relationship held by contemporary workers (Rendenz, von Lanz, Belonoschkin, and Young, loc. cit.).Redenz and Belonoschkin seem to be of the opinion that all internal or protoplasmic changes undergone by spermatozoa during their development, occur while these cells are still attached to the germinal epithelium. These changes include the development of the capacity for independent motion. The spermatozoa are then loosened from the germinal epithelium, and swim by means of their own strength through the rete tubules and vasa efferentia into the ductus epididymidis. At this time the spermatozoa are mature internally, but unprotected externally from the effects of injurious factors in the environment, such as acids and high temperature, which act to decrease the intensity and duration of motion. As they pass through the epididymis, however, they become enclosed in a microscopically invisible protective envelope (Oberflächenhäutchen or Hülle) composed of epididymal secretion. This adherent layer of secretion provides the external protection they require, and thus imparts to them a greater resistance against the action of injurious factors which they may encounter. In other words, Rendenz and Belonoschkin would seem to be of the opinion that the ripeness or maturity attained by spermatozoa during their passage through the epididymis is a quality conditioned by the presence of the epididymal secretion around the spermatozoon, and is not a development inherent in the spermatozoon.An equally important part of their theory concerns the provision made for the preservation of spermatozoon vitality until the time of an ejaculation. This is said to be accomplished by the limitation of the ability for independent motion which spermatozoa contained in the epididymis are assumed to possess, a condition which prevents the dissipation of the energy available for motion in useless movements. This limitation of motion, in turn, is thought to be accomplished by some action of the high carbon dioxide tension and the low oxygen tension which are consequences of the density of the stored spermatozoa. Restoration of the motile condition is believed to be brought about at the time of an ejaculation, possibly by the oxygenation which occurs, and possibly by the alkalinity of the prostatic and seminal vesicle secretions with which the spermatozoa become mixed.In only one place does Redenz (1926, p. 135) suggest that the vitality of spermatozoa may not be preserved indefinitely.Der Begriff "Reifung" hat aber noch einem anderen Inhalt. Nachdem ich zugleich mit v. Lanz mit ganz verschiedenen Methoden feststellen konnte, dass der Nebenhoden ein Organ ist, das lebende Samenfäden speichern kann, ist die Frage, in welchem Zeit-punkt das Spermium nach seiner Entstehung zur Befruchtung kommt, von wesentlicher Bedeutung geworden. Es ist damit zum erstenmal die Frage angeschnitten, welchen Einfluss auf die Bewegungsdauer, auf die Zeugung, ja auch auf die Vererbung und die Geschlechtsbestimmung eine solche Überreife im männlichen Organ hat. Zwar werden Spermien, die sehr lange im Nebenhodenschweif verweilen, höchstwahrscheinlich auch an ihrem Sekretmantel Schaden leiden und damit weniger Aussichten haben, zur Befruchtung zu gelangen. Jedenfalls ist zu der Frage des Eialters, bei der Befruchtung, vom Follikelsprung angerechnet, die des Spermienalters, gemessen von der Abstossung aus der Sertolizelle an, hinzugetreten.This suggestion does not seem to be incorporated in the theory of the function of the epididymis expressed in more recent papers, however, and it is difficult to conclude how much importance he attaches to it.The most recent theory of the epididymal spermatozoon relationship advanced by von Lanz (1929) is similar in many respects to that formulated by Redenz. Like the latter, von Lanz is of the opinion that spermatozoa move out of the testis by means of their own motion, that important ripening changes occur during their passage through the epididymis in consequence of some specific action of the epididymal secretion, and that the preservation of spermatozoon vitality during the residence of these cells in the epididymis is accomplished by the limitation of their motion. Unlike Redenz, he believes that a considerable buffering action of the epididymal secretion must be carried over to the protoplasm of the spermatozoon during storage in the epididymis, that ripening events take place inside the cell protoplasm and not around it, that the limitation of motion is accomplished by the natural acidity of the epididymal secretion rather than by any excess of carbon dioxide or deficiency of oxygen, and in no place does he suggest that spermatozoon vitality may not be preserved indefinitely.Many observations made in the course of experiments performed by the writer are at variance with certain of the more important assumptions and conclusions which are a part of the theories of the epididymal spermatozoon relationship advanced by Redenz and von Lanz. It is doubtful, first of all, on the basis of many observations on testes from bulls and rams (Young, 1929 a), if spermatozoa exhibit an independent motion while they are still contained within the seminiferous tubules, or take any active part in their movement into the epididymis. Only the weakest vibratile movement of the tail has ever been detected by the writer following the maceration of testis fragments in Locke's solution, a flagellation which has never been observed to result in progressive motion. Furthermore, spermatozoa removed from distal parts of the epididymis are activated more quickly than those removed from proximal levels. If spermatozoa do move into the epididymis by means of their own strength, and if their motion is limited gradually by the nature of the environment found within this organ, one would expect that spermatozoa in the distal end of the structure where their motion is most completely limited, would be the last rather than the first to be activated. The nature of the epididymal secretion may be such as to prevent the occurrence of motion, but it probably does not limit motion which once existed.A second point emphasised by Redenz and von Lanz, which is questioned on the basis of data reported previously (Young, 1929 a) and in this paper, is that developmental changes undergone by spermatozoa during their passage through the epididymis are conditioned by some specific action of its secretion. It has been found, for example, that the extent to which spermatozoa removed from the seminiferous tubules can be stimulated to motion varies from species to species. In a preparation from the testis of the rat, hundreds of motile spermatozoa can be seen. In preparations from the testes of the bull, ram, and guinea-pig, on the other hand, only an occasional spermatozoon, which exhibits the weakest vibratile movement of the tail, can be seen. Evidently, in some species under normal conditions, a certain portion of the developmental process occurs only after the spermatozoa have been carried into the epididymis, whereas, in other species, corresponding changes occur while the spermatozoa are still within the testis.It has also been observed that the number of spermatozoa which can be stimulated to motion, following their removal from the testis, is often much greater when the escape of spermatozoa from the testis has been prevented by the ligature of the head of the epididymis than it is when the testis is normal. From this it would seem, that in any given animal, many of the developmental changes which normally occur after the spermatozoa have entered the epididymis will occur while these cells are still contained in the testis, if their passage from the testis into the epididymis is prevented.If the above described observations have been interpreted correctly, the older conception of spermatozoon development as consisting of two processes, one of which occurs in the testis and the other of which occurs only in the epididymis, seems questionable. It is suggested instead, that spermatozoon development is a single continuous process occurring in part in the testis and in part in the epididymis, and that the extent to which development occurs in the testis or in the epididymis depends, not upon one set of stimuli in the testis and upon another set in the epididymis, but rather upon the proportion of the total developmental period spent in each of the two organs.A third criticism of the theories advanced by Redenz and von Lanz is that neither makes proper allowance for the fact noted elsewhere (Young, 1929 b, and others) and in this paper, that regressive as well as developmental changes may occur in spermatozoa during their passage through the epididymis. Inclusion of this fact in any theory of the epididymal spermatozoon relationship is important, not only to complete the account of the post-testicular history of spermatozoa, but also because certain problems are raised which otherwise would be overlooked, and which may prove of some importance for the part played by the male in reproduction.What would appear to be the most important of these centre around the rate of progress of spermatozoa through the epididymis, viz. how much time is required for the passage of spermatozoa through the epididymis? To what extent does this rate of progress vary from male to male? To what extent is it modified by the frequency of copulation and by other factors? Do differences exist in the physiological condition of discharged mammalian spermatozoa similar to those noted by Lillie (1915) and by Goldforb (1929 a, b, c, and earlier papers) in sea-urchin spermatozoa? If such differences exist in mammalian spermatozoa, can they be related to differences in the rate of passage of spermatozoa through the epididymis ? Lastly, if such differences exist in discharged mammalian spermatozoa, are they important for any reproductive qualities of the male mammal?Most of these questions are now being investigated, and it is expected that the answers, when such are obtained, will supplement the theory of the epididymal spermatozoon relationship elaborated in this and the following paper (Simeone and Young, 1931).
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