A Study of the Function of the Epididymis

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

BY WILLIAM C. YOUNG.

(Arnold Biological Laboratory, Brown University.)

(Received zist September, 1930.)

(With One Text-figure.)

INTRODUCTION.

IN 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 develop-mental 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).

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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 spermato-zoon 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 develop-ment which are not different from those found in the testis are present; that sperma-tozoa 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 develop-ment in which these cells are constantly changing; ripening until an optimal func-tional 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).

EXPERIMENTAL.

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.

';

'.:X\£~- '•'y--$'&~ •'•=•'&':??: '•;

Fig. i. Testis, epididymis, and proximal end of vas deferens from guinea-pig as seen when retracted into the abdominal cavity, a.. .b is line of separation of cauda epididymidis into proximal and distal portions from which younger and older spermatozoa respectively were removed from normal males. Lines c, d, and e represent location of ligatures in experimental males.

154 W I L L I A M C. YOUNG

from the distal end of the cauda epididymidis from the same male. The females^ 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 Pe r 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 modifica-tion 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.

Pnto 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 con-tamination 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 ex-pected 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 im-pregnated. 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, there-fore, 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 im-pregnated. Of the females inseminated with spermatozoa from the distal end of the column, twenty-five, or 25 per cent., were impregnated.

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in 68 per cent, of the cases. Further proof came from the fact that the ability or' 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 de-creased 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.

Table I. Summary of data obtained following insemination of females with spermatozoa

removed from proximal and distal ends of normal and ligatured cauda epididymides.

Number of inseminations Fertile inseminations (%) Average litter size Number of normal

Number of still-born indi-viduals

Number of aborted and re-sorbed individuals Still-born individuals (%) Aborted and resorbed

indi-viduals (%)

Normal males (control)

Proximal end 9 9 33'3 1-85 48 7 2 12-3 3 6 Distal end 9 7 6 8 0

2 6 0 139 13 15 7-8 9 0 20-day Proximal end 43 44-2

2 5 0 32 2 10 ± 4 8 22-7 ligature Distal end 4 0 32'5 2-S8

23 2 ± 6 8 2 2O-O 25-day Proximal end 98

4 9 0

2-34 7 2 9 21 ± 9'4 20-9 ligature Distal end

1 0 0

25-0 2-36 43 6 9 ± 10-7 18-8

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 investiga-tion.

I931), 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 develop-ment 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 i n the semini-ferous 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 con-tinued 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 sperma-tozoa 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 pre-sented, 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

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centage of individuals which were aborted or resorbed, on the other hand, 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 fertilisa-tion 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.

DISCUSSION.

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 contem-porary workers (Rendenz, von Lanz, Belonoschkin, and Young, loc. cit.).

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 con-sequences 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 sperma-tozoa 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 Samenfaden 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 Uberreife im mannlichen Organ hat. Zwar werden Spermien, die sehr lange im Nebenhodenschweif verweilen, hochstwahrscheinlich auch an ihrem Sekretmantel Schaden leiden und damit weniger Aussichten haben, zur Be-fruchtung zu gelangen. Jedenfalls ist zu der Frage des Eialters, bei der BeBe-fruchtung, 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.

160 W I L L I A M C. YOUNG

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 semini-ferous 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 stimu-lated 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.

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 repro-duction.

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 physio-logical 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).

CONCLUSIONS.

1. By means of experiments which have involved the artificial insemination of female guinea-pigs with spermatozoa removed from different levels of the ductus epididymidis and observations on spermatozoa confined in the seminiferous tubules, further evidence has been obtained for the conclusion (1) that the time consumed by spermatozoa in passing through the epididymis is necessary for a completion of their development, (2) that the changes undergone during this period represent a continuation of changes which start while the spermatozoa are still attached to the germinal epithelium, and are not conditioned by some specific action of the epididymal secretion, and (3) that in case the spermatozoa are not discharged or lost in some other manner, regressive or degenerative changes follow the attainment of an optimal functional capacity.

162 W I L L I A M C. Y O U N G

of copulations. The importance of determining this rate of progress of spermatozoa through the epididymis is emphasised on account of its possible relationship to the physiological condition of ejaculated spermatozoa.

3. The greater frequency with which abortions and intra-uterine resorptions occurred among females inseminated with older spermatozoa has been noted, as was the fact that several of the foetuses lost in this manner showed gross structural defects.

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BRAUS, H. and REDENZ, E. (1924). Anat. Anz. 58, 121-131. DUNGAY, N. S. (1913). Biol. Bull. 25, 213-261.

GOLDFORB, A. J. (1929 a). Biol. Bull. 57, 333-349. (1929 b). Biol. Bull. 57, 350-388.

(1929 c). Biol. Bull. 57, 389-411.

VON LANZ, T. (1924 a). Verh. d. anat. Gesellsch. 33, 106-115. (1924 b). Zeitschr.f. Anat. u. Ent.-geschichte, 74, 761-815. (1926). Zeitschr.f. Anat. u. Ent.-geschichte, 80, 177-282. (1929). Pfluger's Arch. 222, 181-214.

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(1926). Wurzburger Abhandlungen aus dem Gesamtgebiet der Medizin, Neue Folge, 4, 107-150. (1929). Zeitschr.f. Zellforsch. u. mikrosk. Anat. 9, 734-749.

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