View source for Paper - The histology of the retained testis in the human subject at different ages, and its comparison with the scrotal testis (1929)

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=The Histology of the Retained Testis in the Human Subject at Different Ages, and its Comparison with the Scrotal Testis =

By Eugenia R. A. Cooper, M.D., M.Sc. 

Lecturer in Histology in the University of Manchester

==Introduction==

The object of this research was to compare the structure of the normal and retained testes in childhood, with a view to establishing whether changes in the latter glands were primary or secondary. Moreover, it was hoped that the results would enable one to decide whether or not it was justifiable to advocate transplantation of the retained gland in infancy, with a reasonable prospect of its complete development when placed into its natural environment.


During the past few years, great interest has centred around the ectopic testis in man and animals, and much valuable work has been done by Crewe (6, 7), Bland-Sutton (4), Turner (38), Bevan(2), Moore (21-29), Thorek (35-37), Lipschutz 20), Fukui (11), Hobday (14), Bissonnette (3) and Eccles (9).

It appears to be generally agreed that undescended testes in later life are functionless as regards spermatogenesis. It is still controversial, however, whether the imperfection is primary, or secondary and dependent upon the persistence of the malposition.

==Technique==

The glands examined were all subjected to the same histological technique, so that a standard was maintained, and influences due to different methods were obliterated.


All adhering connective tissue was removed until the tunica albuginea was reached. The glands were cut into three parts, 4, B and C, by two incisions transverse to the long axis, the upper one passing through the centre of the globus major, the lower one through the body of the epididymis. Thus the upper, middle and lower parts of the testis were available for easy examination, and also the three parts of the epididymis.


The retained organs were obtained from the operating theatre and fixed immediately, post-mortem changes being thus overcome. The scrotal glands were necessarily removed at autopsies, but they were fixed as soon as possible, and post-mortem changes were reduced to the. minimum.


The number of retained testes examined was unavoidably small, since, with modern operative technique, the majority of the organs are now transplanted into the scrotum rather than excised. (Twelve retained testes were obtained and a series of 36 scrotal organs was used for comparison.)

All organs were fixed in 10 per cent. aqueous solution of commercial formalin. Fresh sections of Block C were cut by the freezing microtome and stained with Sudan III and haemalum. Blocks 4 and B were embedded in paraffin and all sections cut at 6». Three methods of staining were adopted:

# Haemalum and aqueous eosin, for general observations.
# Mallory’s Saure-fuchsin phospho-molybdie acid stain for connective tissue.
# Heidenhain’s iron haematoxylin method for special cytological observations.

==Observations==

During the last two months of human intra-uterine life the testis is situated somewhere between the posterior abdominal wall and the scrotum, usually completing its descent before birth in a full-time foetus. Normally by the seventh month the testis in the human subject has attained a position at the internal abdominal ring(16), or has entered the inguinal canal. The gland examined at this age was oval in shape and of small size. The epididymis was situated above and posteriorly to the testis and was freely movable, being attached above only by a small rete testis to the upper pole of the gland, and otherwise separated from it by a wide digital fossa, measuring about 0-3 cm. at its widest point. The vas deferens emerged below from the globus minor.

Histologically the organ was surrounded by a delicate fibrous tunica albuginea which projected into the gland at one point, forming the mediastinum or hilum testis. The connective tissue of the hilum was continuous with that of the digital fossa, and from it fibrous strands radiated into the testis forming a fine meshwork supporting the tubules. The supporting tissue throughout was of a delicate nucleated nature, except the more peripheral parts of the tunica albuginea, where more mature fibres were observed. The arrangement of the supporting elements was found to be similar at all subsequent ages, except that as age advanced coarser fibrous tissue replaced the delicate nucleated material. No further reference will be made to this point.

At this age the testis consisted of small tubules measuring about 42 in diameter (PI. I, fig. 11). They were loosely arranged, the intervening intervals being filled by delicate fibres. There was no definite fibrous basement membrane, but the nucleated connective tissue was condensed around each tube. The tubes were entirely filled by closely packed seminiferous cells, the majority presenting the same appearance—-a round, pale, oval nucleus, exhibiting definite nuclear membrane and spots, embedded in an ill-defined mass of cytoplasm. The nuclear spots, arranged irregularly within the membrane, represented the chromatin reticulum. These cells are best described as undifferentiated germinal epithelium cells(19). The nuclei of these cells present differences in their affinity for stains. It is probable that one type would differentiate into cells of Sertoli, and the other into spermatogonia. At this age no definite cells of Sertoli were present.


1 Diameters represent the mean taken from measurement of 10 circular tubules in true transverse section. 



Frequently, with lower microscopic powers, some cells seemed to have “escaped” from the tubules, but this appearance was due to the accumulation of the nuclei of the delicate stroma round the tubes.

In many of the tubes, one or more larger cells were present, consisting of a large, round, deeply stained nucleus lying in the middle of a big, round cell body, of which the cytoplasm was sometimes pale and ragged, but often definite and well stained. The nuclear membrane was distinct, and several dark nuclear spots, representing the chromatin, were arranged just inside the membrane. These cells were differentiated spermatogonia and were always present in the peripheral parts of the tube (PI. I, fig. 1).

Throughout the testis, in the intertubular tissue, small accumulations of large polygonal cells were observed. The protoplasm was finely vacuolated, presumably due to the removal of fatty material in the course of the histological preparation. The cell outlines were often indistinct and the mass appeared syncytial. The nuclei were well stained and larger in size than any of those of the tubular cells, and each contained usually two nucleoli. These cells were the interstitial cells of Leydig and were present in large numbers in all parts of the gland. They presented a similar appearance throughout life.

In section, the epididymis was of the same size as the testis. It consisted of round, empty tubes lined by a single layer of ciliated columnar epithelium. The tubes were isolated by delicate fibrous tissue continuous with that of the digital fossa, and more compact immediately around each tube. No further reference will be made to the epididymis, as no alterations, beyond increase in the number and size of tubules, were observed.

The earliest age at which the retained testis was examined was fourteen months, and this gland was situated’ in the right inguinal canal near the external abdominal ring. It was approximately equal in size to the normal organs of the same age. The digital fossa was open, an interval of 0-45 cm. separating the body of the testis and the epididymis, whilst a stalked appendix (vas aberrans) was suspended from the lower pole of the testis (PI. I, fig. 2 a).

Normal glands examined during the first two years of post-natal life often presented an open digital fossa, and a vas aberrans was not rare (PI. I, fig. 2B). It was repeatedly noticed, in both scrotal and retained testes, that a persistent digital fossa was frequently accompanied by a vas aberrans.

In sections of this retained testis a tunica albuginea of moderate width surrounded the organ. The tunica, mediastinum testis, and digital fossa all consisted of delicate fibrous tissue. The tubules were closely packed and equal in size to those of the scrotal glands of the same age, measuring 65 p» in transverse section. Each tube was enveloped in a well-defined basement membrane. 



In the centre of the gland, coarser fibrous tissue separated the tubes, indicating a commencing fibrosis (Pl. I, fig. 3B). The greater number of tubules were filled with cells, but there was a tendency for them to become arranged in rows against the basement membrane, the centre being empty or containing more loosely arranged cells. Most of the cells had a similar appearance, and were identical with the undifferentiated cells of scrotal testes (Pl. I, fig. 3 a).

As in the normal testis, the nuclei showed a differential staining. One or two larger cells were present in each tube, resting on the basement membrane. These were the spermatogonia, and were in all respects similar to those of scrotal glands.

Here and there a small group of interstitial cells was observed in the intertubular intervals. The epididymis in all parts was characteristic in appearance.

Towards the end of the second year the scrotal testes examined were found to vary considerably, both anatomically and histologically. Some were characterised by the patent digital fossa and the presence of a vas aberrans. In others the testis and epididymis were in contact and there was no appendage.

Histologically, glands of the former series showed a reversion to the earlier condition. The supporting tissue was embryonic. The tubes were isolated and devoid of a true basement membrane. They were of small size, measuring 46 » in section. Two or more spermatogonia were present in each tube. The rest of the cells were undifferentiated, often presenting a syncytial appearance. There were no interstitial cells.

Testes of the second series were in all respects similar to the glands described as characteristic for this period. The supporting elements were truly fibrous, and a definite fibrous basement membrane surrounded each tube. The tubes were closely packed and measured 50y. They were filled with undifferentiated cells amongst which were two or more spermatogonia. In the latter were some very large nuclei probably preparing to undergo division in situ (Pl. I, fig. 3 4).

Thus it will be seen that the retained testis of fourteen months was a compromise between two types of scrotal organs occurring at this age. Whilst the histological elements conformed to the type characteristic of this period, the anatomical details were similar to those of scrotal glands of younger age. The slight thickening of the intertubular connective tissue in the retained organ was the only distinguishing feature.

The retained testis obtained from a child of two and a half years was situated high up in the right inguinal canal. Its dimensions were all smaller than those of a completely descended testis of the same age. It was accompanied by a large patent processus vaginalis. The epididymis was curious. It was in close contact with the testis posteriorly, the digital fossa being almost closed. Above, it was only loosely attached to the upper pole of the gland, and was then prolonged downwards, hanging freely over the anterior margin of the testis. Histologically this testis was enclosed in a delicate fibrous tunica albuginea of similar width to that of the scrotal gland of the same age. The tubes were scattered irregularly throughout the gland; at the periphery they were closer together, whilst in the centre they occurred in small groups widely separated by delicate nucleated supporting tissue. There was the appearance of cells having “escaped” from the tubes, but on closer examination these cells were seen to be part of the nucleated intertubular stroma which was condensed in the vicinity of the tubes. This feature was observed in the foetal testis (Pl. I, fig. 4).

There was evidence of a commencing fibrosis in the centre of the gland, but so far the tissue was of a delicate texture. The tubules were of small size, measuring in transverse section 57. Each was enclosed within a fine but definite basement membrane. The tubes were more or less filled with cells, the majority of which were undifferentiated. epithelial cells. In the minority of tubes, definite spermatogonia were present, two or three or more occurring in one tubule. On the whole the spermatogonia were proportionately less numerous than in the scrotal testis of the same age, even considering the reduction in the number of tubules in this retained’ gland. The spermatogonia present, however, were similar in all respects to those in completely descended glands. An occasional small group of interstitial cells was recognised, possibly indicating that the organ had developed more slowly than was normal.


Scrotal testes in the third year again presented anatomical variations. In a few, there was an open digital fossa and a vas aberrans. In the remainder, the fossa was closed and there was no appendage. The tubules were closely approximated throughout the organ, being separated only by their basement membranes. They measured 62» in diameter. Three or more large spermatogonia were present in each tube projecting into the lumen amongst the undifferentiated cells.


Comparing the retained testis from a child of two and a half years with scrotal ones of the same age, it will be seen that there were distinct alterations. There was a reduction in the number and size of the tubules, and an increase in the intervening stroma. The tubular cells, however, were characteristic in appearance though not in numbers.

A testis retained at the internal abdominal ring was obtained from.a child of four years. It was similar in size to a scrotal gland of a child aged five and a half years. The globus major and minor of the epididymis were in contact with the upper and lower poles of the testis respectively, but the body was separated from the posterior margin of the testis by a wide digital fossa measuring 0-5 cm. at its widest point. A tiny vas aberrans was suspended frem the lower end of the testis.


In the centre of the gland there was a considerable thickening of fibrous tissue, and amongst it were small isolated tubes, each supported by a delicate fibrous basement membrane. At the periphery, the tubes were similar in arrangement, but fine nucleated connective tissue occupied the intertubular intervals. The diameter of the tubes was 46. Most of them contained a single or double layer of cells lying inside the basement membrane, but some were filled by cells. The cells were undifferentiated, the nuclei again presenting a differential staining. The limits of the protoplasm of each cell were ill-defined. The majority of tubules contained one or more typical spermatogonia. In a few, a double nucleus was observed. No interstitial cells were seen in this testis.


Another testis retained in the higher part of the inguinal canal of a child of four and a half years was examined. In size this gland was similar to the one described above. The digital fossa was closed. Histologically the organ consisted of small tubes scattered evenly throughout the gland. The intertubular tissue in all parts was of a fine nucleated character, forming concentric rings round each tubule, the basement membrane not being definitely formed. In transverse section the tubes measured 38p. Each contained closely packed undifferentiated cells lying in rows at the periphery of the tube. Many of the tubes contained one or more large characteristic spermatogonia. No interstitial cells were recognisable in this gland. The epididymis ealled for no special description.

Another testis retained at the internal abdominal ring in a child of five years was obtained. The tunica albuginea was wide and composed of very delicate nucleated stroma. The tubules measured 38 », but were not numerous (PL. I, fig. 5). They were irregularly arranged in groups throughout the organ, and delicate fibrous tissue occupied the intervals between them, becoming more concentrated around each tube. There was no distinct basement membrane. Typical undifferentiated cells were gathered into two or three rows at the periphery of the tubes, the centre being empty. Some tubes contained two, three or four spermatogonia resting against the basement membrane amongst the undifferentiated cells. The spermatogonia were of large size and characteristic in structure. The scrotal testes examined during the fifth year were found to be comparatively small in size, showing no increase on those ‘of the fourth year. The digital fossa was closed in all cases. The tubules were closely approximated throughout the organ, and were small, measuring 50 on an average. They contained undifferentiated cells, usually accumulated near the basement membrane. Spermatogonia were present. Some of them contained a double nucleus, probably resulting from recent division. Interstitial cells were absent. Comparing the retained testes obtained from children between the ages of four and five years with scrotal ones, it will be seen that in the former there was a great reduction in the number of tubules. The variation in size was not so apparent, since in the normal glands the size was variable. The fibrosis in the centre was confined to the retained organs. Although the tubular cells were collectively reduced in the latter, their histological structure was characteristic.

An undescended gland obtained from a child of six years was abdominal in situation. It was of larger dimensions than scrotal glands of that age, and all parts of the epididymis were removed from the testis (Pl. II, fig. 6 a). 


digital fossa between the body of the epididymis and the testis measured 1-4cm. A vas aberrans was suspended by a long stalk from the lower end of the epididymis. A wide fibrous tunica albuginea surrounded the organ, and dense fibres invaded the peripheral parts of the testis. Within the tunica the interstitial tissue was of a delicate nucleated nature, and immediately around each tube became more compact, forming three or four concentric rings. The basement membrane was not distinctly formed.


The tubés were of small size and scattered unevenly (PI. II, fig. 68). In transverse section they measured 46. At the periphery of the testis and near the hilum the tubules were more numerous, but in the central parts they were separated by wide intervals occupied by a fine meshwork of fibres. The tubes contained irregularly arranged cells, the majority of which were undifferentiated. Some of the tubes, especially those near the tunica, contained one or more larger cells, similar in all details to spermatogonia. Although they were absent in most of the tubes, yet as many as five were counted in a single tube.


No cells of Leydig were observed in the intertubular tissue. The epididymis was in no way different from that of scrotal testes. Unfortunately scrotal testes from children between the ages of six and seven were not obtained.


One examined from a child of eight years showed a reversion to an earlier type (PI. II, fig. 7). It was composed of scattered tubes of small size measuring 50 p in section. The intertubular tissue was of an infantile, nucleated character, though more mature fibres formed the basement membranes. The tubes were irregularly distributed, being in closer contact near the mediastinum than elsewhere.


Undifferentiated cells were arranged in the peripheral parts of the tubes, the centre being empty. Three or four or more spermatogonia were present in each tube, many showing signs of nuclear division in situ. As yet no cells of Sertoli as described by Winiwarter (41) and Allen(1) were distinguishable.


Comparison of the abdominal retained testis of a child of six years with a scrotal gland of a child of eight years, showed that a reversion to a more primitive type (characterised by a smaller number and size of the tubes and a greater amount of interstitial stroma) is not confined to the retained gland, and may occur even in scrotal organs.


No retained testes were available between the ages of seven and twelve years. The normal glands examined during this period were found to increase greatly in size, attaining adult dimensions about the thirteenth year (PI. IT, fig. 9). The tubules in these cases were closely packed, separated only by their basement membranes. In one gland from a child of eleven years, there was a persistent digital fossa and a vas aberrans. The tubules in this case measured only 50, and were again separated, the intervals being occupied by delicate stroma. Two or more spermatogonia were present in each tube, some containing a large nucleus, and others a double nucleus resulting from recent division. At this period occasional interstitial cells were recognisable in the intertubular spaces. 


In all the organs examined from the tenth year onwards to puberty, a noticeable change occurred in some of the undifferentiated cells. Instead of the innumerable chromatin spots in all parts of the nucleus, as in the true undifferentiated cells, the background of the nucleus was pale, and one to four definite nucleolar spots of variable size lay irregularly inside the nuclear membrane. They occurred either separately or in pairs, near the periphery of the nucleus or in the centre. The protoplasm was of a fine reticulated nature, indistinguishable from that of the ordinary undifferentiated cells, being syncytial in both cases as far as could be ascertained in post-mortem material. The nuclei of some of these differentiated cells corresponded very closely to those described as characteristic of cells of Sertoli by Regaud(34), Winiwarter (41) and Allen(1). It is probable that this nuclear differentiation indicated a further stage in the development of cells of Sertoli.

An imperfectly descended testis was obtained from the right inguinal canal of a child of thirteen years. In size it corresponded to scrotal glands of the same age. Unlike these organs, however, the digital fossa was still open, the body of the testis and epididymis being separated by an interval of 08cm. A large vas aberrans was suspended by a long stalk from the globus minor.

Histologically a fibrous tunica albuginea of normal width surrounded the testis. The parenchyma of the gland consisted of tubules of similar size to those of a scrotal testis at puberty, measuring 92» in diameter (PI. IT, fig. 8). Each was enclosed within a well-defined basement membrane, but the intertubular intervals were occupied by delicate connective tissue fibres. The tubes were in contact with one another in the peripheral parts of the testis, but were more widely separated internally. They contained cells lying in two or three layers against the basement membrane, leaving an empty central lumen. Three types of cells were recognisable:

# Many were of the undifferentiated type.
# Large characteristic spermatogonia.
# Many cells were differentiated into characteristic cells of Sertoli.


The undifferentiated cells usually occurred against the basement membrane, while the differentiated ones were more internal.

A striking feature of the tubes of this gland was the large number of big spermatogonia present in each. Often the small tubular cells were hidden by the germ cells (Pl. IT, fig. 8).

Numerous groups of interstitial cells were seen in the intertubular tissue. Compared with scrotal glands of this age, these cells did not seem to be in excessive numbers. The epididymis was histologically similar in all respects to that of the ordinary adolescent glands.

Another retained gland, from a subject of thirteen years, was of extraordinarily large size. It was abdominal in situation. As in some of the other retained testes, the digital fossa was open, and measured 1-5 cm. approximately in width. A large stalked appendix was suspended from the lower pole of the testis where it lay in contact with the globus minor.


In section this testis consisted almost entirely of fibrous tissue. A very wide tunica albuginea enveloped the organ, but was of a coarser fibrous nature than that in the centre. The limit of the tunica was recognised by the change in the character of the fibrous tissue, and by the presence of a ring of tubules arranged in small groups round the periphery of the gland. Here they were closely approximated, but the central parts contained only an occasional tube. The tubes were surrounded by a definite basement membrane, and they presented a peripheral accumulation of cells. They measured 49 u in transverse section. The majority of cells were specialised forms of undifferentiated cells, and corresponded to those described as cells of Sertoli. Only a few typical spermatogonia were recognisable, and were not present in the majority of the tubes. In the interstitial tissue between the tubes, groups of typical cells of Leydig were seen. It was significant to find that they occurred only in the intertubular regions of the gland. The epididymis called for no special description.

A scrotal testis obtained from a child of twelve years was of large size, and anatomically similar to the fully developed organ (PI. II, fig. 9). The tubules of the gland measured 90» in diameter, and throughout the organ were evenly distributed and in close contact with one another. Each was enveloped by a fibrous basement membrane. The tubules all exhibited an irregular empty central lumen, with two, three or four layers of cells around it. Large, typical spermatogonia were present in all the tubes, resting against the basement membrane. Many showed a double nucleus, the result of recent division, and the newly formed spermatogonia were smaller in size and contained a small, darkly stained nucleus.

Active nuclear division of the spermatogonia was visible in the majority of tubes. Spermatocytes were differentiated, and their nuclei showed all stages of fragmentation. Spermatids and spermatozoa were not recognisable. A large number of undifferentiated cells had by this age undergone complete differentiation into cells of Sertoli, characterised by their large pale nuclei and cell body of ill-defined limits, in contrast to the active nuclei of the germjnal cells. Many interstitial cells were present in groups in the intertubular angles.


At thirteen years the testes examined were approaching adult dimensions. The digital fossa was “closed,” although the thin outer margin of the epididymis could be raised from the testis and a finger inserted into the fossa. The tubes were of large size measuring 114 in transverse section, and each was separated from its neighbours by its basement membrane. Spermatogonia were present in the tubes resting against the basement membrane. More internally were the spermatocytes, the nuclei of which presented all stages of mitotic division. An additional stage in the development of the germinal cells was seen in some of the tubes. This consisted of a small, round or oval, darkly stained nucleus in a small amount of cytoplasm. These were spermatids, and were always near the centre of the tubule. As yet there were no spermatozoa. 


As in the testis of twelve years, differentiation of the small’ primitive cells was indicated by the change in the nucleus, which exhibited two distinct nucleoli, instead of a “‘véritable poussiére” of chromatin spots which characterised the early undifferentiated cells. In many tubes the mitotic changes in the germinal cell nuclei were not apparent, and in those cases the differentiated forms of the undifferentiated cells were very obvious. The arrangement of the protoplasm and the distinctive nucleus rendered these cells indistinguishable from the cells of Sertoli. Regaud(34) and Allen(1) state that they never observed true cells of Sertoli until spermatids had formed.

Another type of scrotal testis was found at the thirteenth year. Histologically it belonged to an earlier type. The tubes were of comparatively small but variable size, and were in contact. They contained closely packed cells amongst which were spermatogonia resting against the basement membrane. These cells showed variations in their nuclei, some being large in size and ready to divide, others smaller and probably resting. Only occasionally was there any mitotic change in the germinal cells. The other tubular cells were either undifferentiated, or definite cells of Sertoli. Groups of large syncytial interstitial cells were present in the intertubular angles in all parts of the organ.


Thus a testis retained in the lower part of the inguinal canal in a boy of thirteen years presented many features in common with a scrotal one of the same age. For instance, in both cases, cells of Sertoli were differentiated, but, on the other hand, mitotic changes in the germinal cells were not observed. The testis of this age retained in the abdomen showed marked fibrosis. In this gland and in the one retained in the inguinal canal the three types of tubular cells (undifferentiated, spermatogonia and cells of Sertoli) were present and similar to those of the scrotal organs, though perhaps reduced in number.


A retained testis was obtained from a person of fifteen years. It was situated just inside the left internal abdominal ring, and was accompanied by a patent processus vaginalis. It was of large size, and the epididymis was separated from the testis by a wide interval, occupied by fibrous tissue. The organ was surrounded by a thickened fibrous tunica albuginea, and was composed of large tubes measuring 121 » in diameter, all isolated by delicate, vascular stroma, and surrounded by a well-marked fibrous basement membrane. The tubes were not characteristic of the age, for they contained comparatively few cells, showing no nuclear activity (Pl. II, fig. 10). They were aggregated in two or three rows against the basement membrane, whilst the large central lumen was occupied by strands of protoplasm prolonged from that surrounding the nuclei. In some of the smaller tubes, loosely arranged syncytial cells filled the lumen.


The cells were in many cases degenerated, but some were characteristic cells of Sertoli. Only an occasional spermatogonium was seen, but those present were in the resting condition. Large groups of interstitial cells occupied the intertubular intervals. These cells appeared to be in excessive numbers, but compared with scrotal glands of the same age, this richness was only apparent, the cells of Leydig being more conspicuous on account: of the wide separation of the tubes. '


Scrotal testes examined at puberty between the fourteenth and sixteenth years had assumed adult characteristics. The tubules in these cases were of large size, measuring 137 » in diameter in transverse section. They were closely approximated and evenly distributed throughout the gland. Each was enclosed within a basement membrane. Between the tubules there was finer connective tissue in which groups of interstitial cells were discerned.


Active spermatogenesis was observed in many of the tubes, whilst others appeared to be in a resting condition. Next to the basement, membrane were spermatogonia alternating irregularly with definite cells of Sertoli, which exhibited the characteristic nucleus and nucleolus. The cytoplasm of the cells of Sertoli was syncytial and vacuolated, large round empty spaces occurring irregularly within it. The protoplasm of the spermatogonia, however, was usually of clearly defined limits, but often overlapped and obscured by that of adjacent cells of Sertoli. Simple undifferentiated cells were frequently seen resting immediately against the basement membrane.

Nearer the centre of the tubes were the spermatocytes, the nuclei of which showed different stages of fragmentation. In the most internal parts were spermatids, usually in small groups. They were distinguished by their small, round, well-stained nucleus and well-defined mass of protoplasm. Often they formed an isolated mass in the lumen of the tube.

Lastly, spermatozoa were present, consisting of a lance-shaped head and filamentous tail. Many appeared to Have lost their tails, possibly a result of post-mortem autolysis. As a rule, different stages of spermatogenesis were seen in different parts of the same tube, and usually the spermatozoa, with their heads buried in the syncytial cytoplasm of Sertoli cells, were accumulated in parts where only the nurse cells were present.

Small groups of typical interstitial cells were recognised in the intertubular angles.

The retained testis examined at puberty showed marked changes including the absence of spermatogenesis which normally characterises this period.

A retained testis was obtained from a dissecting-room subject aged sixty years. The gland was equal in size to the right scrotal gland of the same subject. The retained testis was situated on the front of the pubes at the external abdominal ring, and it was fixed to the posterior wall of the scrotum by a strong strand of connective tissue. The globus major surmounted the upper pole of the testis, whilst the body and globus minor of the epididymis lay across the centre of the left aspect of the organ, the vas deferens being posterior.

Microscopically the testis was found to be enclosed within a very wide tunica albuginea composed of tough fibrous strands. Many tubules were present, some of which were healthy, though many were atrophied. Long columns of healthy, active tubules alternated with degenerated tubes or with parallel strands of vascular fibrous tissue. The atrophied tubes were elongated. and narrow, and were surrounded by a delicate basement membrane. Within the tubes were cellular remnants and a few odd nuclei, whilst very fine fibres formed a meshwork in which the cell débris and nuclei were enmeshed. The healthy tubes were elongated and of a large size, and each was surrounded by a greatly thickened basement membrane (text-fig. 1). The tubes were occupied by germinal cells, showing all stages in their maturation. In many tubes definite spermatozoa were recognised by their elongated, very deeply stained heads. They were usually buried in the vacuolated protoplasm of a cell of Sertoli. The tails of the spermatozoa were not recognisable, on account of post-mortem autolysis, since the statutory four days elapsed after death before the body was injected with preservative fluid.




Text-fig. 1. Tubules of testis retained at external abdominal ring, age 60 years. A, spermatogonia. B, spermatocytes. C, spermatids. D, spermatozoa. x 350.


Small groups of polygonal interstitial cells were present in the spaces between both healthy and degenerated tubes.

It was interesting to trace the course of the duct of this retained testis, as, according to Bland-Sutton(4), no account has been given of the vas of retained testes in cases where otherwise the genitalia were normal. It so happened that in this instance, the vas on both sides took an abnormal course. Having passed down the side wall of the pelvis to the ischial spine, it turned acutely inwards to gain the upper border of the seminal vesicle, passing underneath the ureter, which was sharply bent forwards at the ischial spine to enter the bladder. Otherwise the course and termination of the vas were similar to normal cases.


I am indebted to Prof. West of the University of Cardiff for sections of a retained testis of a subject of thirty-eight years. The small glandular mass was removed from the inguinal canal during the operation for radical cure of an inguinal hernia. The patient was a hermaphrodite, presenting a condition of hypospadias, and had lived as a female subject.


The organ was rounded and measured 4mm. in diameter. It was surrounded by a thick fibrous tunica albuginea from which very vascular strands of connective tissue entered the gland. The testis consisted of small scattered tubes, measuring 114 in diameter, each surrounded by a thickened fibrous basement membrane. In places the tubes were widely separated by large vascular strands, or by masses of interstitial cells. Inside the tubes were a few cells, irregularly arranged, some against the basement membrane, others projecting into the cavity, and others lying freely within the lumen of the tube. The cells, with two exceptions, in half a dozen sections examined, presented a similar appearance. Each consisted of an oval or rounded nucleus, of a homogeneous staining reaction, containing usually one large dark nucleolus and frequently a smaller one. As a rule a small amount of fine protoplasm surrounded the nucleus and there was no distinction between the protoplasm of adjacent cells. Judging from the histological condition of the nucleus, it seems justifiable to consider these cells as cells of Sertoli.

In one tubule, one cell of a different kind was observed amongst the cells of Sertoli. It consisted of a well-stained, round nucleus, with a sharply defined nuclear membrane, against which were arranged several small nuclear spots, whilst the central part of the nucleus was paler. Around the nucleus was a small amount of reticulated protoplasm. Compared with the tubular cells in the normal testis, it seems possible that this cell was a spermatogonium (text-fig. 2 a).

In another tubule a cell of large size was seen, in marked contrast to the cells of Sertoli present around it. It consisted of a big, round nucleus surrounded by a well-defined round mass of clear protoplasm. The nucleus was delicately | stained and its chromatin reticulum was in the form of a long filament folded several times on itself. It seems reasonable to consider this cell as a spermatogonium attempting to undergo nuclear activity (text-fig. 2 B).

Large syncytial masses of typical interstitial cells occurred throughout the section. The epididymis was of similar size to the testis in section and its structure was in no way different from the normal, except that the tubes contained no testicular secretion.


Text-fig. 24. Testis of a hermaphrodite of 38 years. A, nucleus of a spermatogonium. B, nucleus of a cell of Sertoli with characteristic nucleolus. x 500.



Text-fig. 23. Testis of a hermaphrodite of 38 years. A, nucleus of a spermatogonium apparently undergoing division. x 500.


==Summary==

From a survey of the findings in the scrotal testes of different ages in childhood, it appears that although the testis increases in size progressively, the development of the organ may sometimes be retarded. This arrest of development is characterised by the isolation of the tubules, the increase of the interstitial tissue which appears in the form of embryonic nucleated stroma, and the diminution in the size of the tubes. The germ cells, or spermatogonia, seem in some cases to be correspondingly reduced in number. Sometimes the digital fossa is open as in foetal glands.

Looking at these details from another point of view, it is suggested that there may: be no arrest in the formation of the tubes, but that the organ is growing in bulk by an increase in the amount of embryonic stroma, and the formation of new and smaller tubes from pre-existing ones. The increase in stroma occurs to accommodate the newly formed tubes which, in their turn, will become larger, on account of the proliferation of the tubular cells, and will ultimately come to be in contact with one another. Mott(31) describes a similar arrest of development during the third year, but in this present research the gland at this age appeared to be growing rapidly.

From foetal life until the tenth year only two types of cells are present in the tubes—spermatogonia and undifferentiated cells. It is believed that the former are developed from the latter, although with the histological methods available, no stages in the transformation could be recognised. In nearly all the glands examined, however, there was a differential staining of the nuclei of the undifferentiated cells, suggesting that one type may ultimately become spermatogonia, and the other develop into true sustentacular cells. It seems certain, however, that spermatogonia are able to divide in situ and reproduce like cells.

From the tenth year onwards, a further differentiation of the undifferentiated cells is clearly recognisable. The characteristic nucleus with innumerable chromatin spots becomes replaced by a paler one with one or two distinctive nucleoli. A similar nucleus in syncytial protoplasm is described as characteristic of the cells of Sertoli by Winiwarter(41), Allen(@) and Regaud (34). At the twelfth year, when spermatogenesis is proceeding, it seems justifiable to regard the specialised undifferentiated cells as true cells of Sertoli, as they are similar in all respects to those occurring in testes where spermatids are present. Regaud(34), whose views are supported by Allen(), states that the first appearance of the nuclei of Sertoli cells in the seminal epithelium coincides, in the rat, with the differentiation of spermatids.

In a gland at the thirteenth year, examined in the course of this research, where spermatogenesis had not commenced, the Sertoli type of nucleus was clearly observed in all the tubes. In all cases, the protoplasm of purely undifferentiated cells, of the specialised undifferentiated cells, and of true cells of Sertoli, was syncytial as far as could be ascertained. Allen(1) and Regaud (34) describe the Sertoli tissue as syncytial, but Winiwarter (41) states that by studying serial sections, he was forced to conclude that the cells of Sertoli were not syncytial, since he could demonstrate a well-defined base and long cell body. This author and Regaud(34) believe that the cells of Sertoli and spermatogonia come from a common source. The results of the present research also point to the same conclusion. Regaud (34), however, was unable to find any transition forms during the differentiation of these two elements from their parent stock, and Winiwarter (41) never found any traces of nuclear division in the adult cells of Sertoli.

The appearance and disappearance of the interstitial cells is a remarkable feature in the human testis. They are present in large numbers during foetal life and until the fourth month after birth. Then, with close approximation of the tubes, the interstitial cells practically disappear and remain in abeyance until the tenth year. They then reappear and gradually increase until possibly their maximum numbers are attained at puberty, after which time they persist throughout life. Mott(31) found numerous interstitial cells in the testis of a newly born child, but at the fourth month they were decreasing rapidly. Whatever their origin, it is interesting to note that, between the ages of five months and ten years, few or no cells with any resemblance to interstitial cells are recognisable in the interstitial tissue.

Apparently the cells of Leydig are influenced by factors other than normal physiological phenomena of birth and puberty, for certain workers (11, 18, 24, 39) describe a hyperplasia of these cells during various experimental investigations on the testis in animals. It appears that the cells of Leydig are an important source of lipoid material in the human subject. The cortex of the suprarenal gland is another source of lipoid, and it is significant to note that it is most active in the production of lipoids during early post-natal life and at puberty (5), probably necessitated by the rapid development of the brain at the former period, and of the maturation of the sexual glands at adolescence. There is, in both the suprarenal and testis, a phase of comparative arrest of lipoid production between these two periods. At present it is not possible to state how far the two organs are associated with one another in this lipoid-producing function, or to what extent they are dependent on each other. 

==Discussion and Conclusions==

From a study of the retained testis in childhood at different ages and in different situations, and comparison with scrotal testes of similar ages, it seems justifiable to draw the following conclusions:

1. The further the pre-adolescent testis has descended in its normal route,

the more closely does it correspond, histologically, to the scrotal gland of the same age.

This conclusion is similar to that of Moore (23), who remarks, after experimental elevation in animals of the testis into the abdomen and later causing its return to the scrotum: “In general it may be stated that the more nearly a testis returns to its normal scrotal position, the more nearly normal will be the tubular epithelium.” Hunter(15) believed that the abdominal retained testis was probably more defective than those late in getting into the scrotum.


It appears from recent work by Crew(é6), Fukui(11), Moore (21-29) and his co-workers, and Bissonnette(3), that the all-important factor for the full development of the testis and maturation of the spermatozoa, is the presence of the gland in the scrotum. Fukui(1l), Crew(6) and Moore(21-29) seem to have proved conclusively that the low temperature of the scrotum is essential for complete spermatogenesis. Thorek (37) has stated that the young ectopic testis is not seriously compromised, and that transplantation into the scrotum has been followed by good functional results. Bissonnette(3), after examination of the high-flanker testis in cattle, concludes that “only descent into the scrotum, either natural or artificial, or other means of cooling the organ, appears to make spermatogenesis possible.” According to Moore (23), “the scrotum exerts a peculiar influence upon the testis, and an influence which is necessary not only for the complete differentiation of the germ cells, but for the retention of cells previously differentiated.”

If the position of the gland in the scrotum is the all essential factor, then, realising that the condition of the retained testis in childhood is healthy, it is justifiable to surmise that such organs will complete their development if placed artificially into the scrotum.

In this research, one gland found in a man of sixty years at the external abdominal ring showed in parts complete spermatogenesis; two others found in the lower part of the inguinal canal in children aged fourteen months and thirteen years, showed features characteristic of pre-adolescent scrotal glands; others situated in the upper part of the inguinal canal presented a reduction in the amount of parenchyma, but a healthy condition of the existing essential elements; other testes occurring in the abdomen were affected by different degrees of fibrosis, and a greater reduction in the germinal elements, but amongst those persisting, a few healthy forms were present.


Hobday (14) states that it is not rare to find spermatozoa in testes which have remained in the lower part of the inguinal canal, but in those found in the upper part or in the abdomen this is exceptional. One case of complete spermatogenesis, including spermatozoa formation, was found by BlandSutton (4), who believes that the retention of a testis is due to its imperfection (vide Hunter (15)): Bland-Sutton (4) also states that a healthy testis is never found in the inguinal canal. It appears from this investigation, however, that a retained testis is not necessarily imperfect, but may present a retarded, but healthy, degree of development.


2. At whatever position the testis is arrested, and at whatever preadolescent age it is examined, the essential elements of the gland, i.e. spermatogonia and undifferentiated cells, are, if not throughout the gland, certainly in many parts, identical in position, structure and relationship with those of scrotal testes of the same age.

This statement corresponds to that of Curling(8) who found the undescended testis healthy but undeveloped.

Despite any reduction in the actual amount of parenchyma, the development and structure of the cells is normal in the retained gland in childhood. Recently Turner (38) has written that full development of the retained testis may be possible up to puberty. Godard (12) believed that the parenchyma of a retained testis was normal, and Félizet and Branca(10) concluded that in a child the lining epithelium of the tubes of undescended testes did not appear very much different from normal.

8. The younger the age at which a retained gland is examined, the more are the normal appearances and characteristics retained.

Rawling (33) has stated recently that in the majority of imperfectly descended testes under ten years of age which he examined, there was no appreciable alteration in the size and consistence of the gland, but in those examined after puberty, it could readily be seen that the organ had not undergone puberty changes. Bland-Sutton(4) has suggested that if a retained testis is functional, it is only for a few years succeeding adolescence. Odiorne and Simmonds (32) state: “It is also true that some testes though undescended, are of normal structure during childhood, but undergo none of the normal changes at puberty.”


From the present examination of retained testes it may be stated conclusively that until puberty many of the tubular cells retain their healthy pre-adolescent characteristics, despite any reduction in the amount of parenchyma. After that time degenerative changes may occur which are more marked according to the higher position in which the gland is arrested. It is essential to add therefore that a retained testis, which is placed artificially into the scrotum, will have a much better chance of completing its development if transplanted at an early age.


Turner (38) suggests that full development of a retained testis may be possible until puberty, after which time pathological processes lead to degeneration. “If the testis be transplanted into the scrotum before such changes occur, there may be a hope that the spermatogenetic function is not entirely lost”’ (Turner (38)). Thorek (37) advocates the transference of ectopic testes into the scrotum before puberty, before atrophy sets in.

Finally it may be said that the changes in the retained testis are secondary. Such a gland develops normally during the early part of post-natal life, but later, if it has not by that time reached the scrotum, then and then only does it undergo degenerative changes.

4, Certain alterations in the imperfectly descended testes occur:

(a) The appearance of fibrous tissue which at first is of a primitive nucleated nature, but later may become fibrosed: this is probably the first change.

(6) “Apparent” disappearance of the tubules, probably due to the increase in the stroma causing the tubules to become more widely separated.

(c) Persistence of the quiescent state of the nuclei of the spermatogonia at puberty.

(d) Disappearance in later years of the more central rows of the tubular cells.

These alterations are recognisable as puberty approaches, and are most marked in those glands which are retained in the abdomen or near the internal abdominal ring. In spite of these changes, however, healthy spermatogonia, undifferentiated cells and cells of Sertoli may be present.

Moore (23) expresses the view that it is known that retained testes are devoid of germinal epithelium, excepting possibly a few spermatogonia. It would appear, however, that so long as even a “few” spermatogonia are present, if the gland be placed into compatible surroundings, they will attempt to mature.

Hanes (13) states that in young pigs, large clear primary sperm cells are seen, but they do not develop further, and no trace of sperm-forming cells can be found in cryptorchid testes in adult pigs.

5. Throughout pre-adolescent life the undifferentiated tubular cells in the retained testis apparently retain their vitality. Many continue to develop into true spermatogonia, whilst others become cells of Sertoli. In both scrotal and retained testes the undifferentiated cells are similar, and at the same period of life (just before puberty) many undergo further specialisation into cells of Sertoli. These are regarded as the nutritive elements of the spermatogenetic cells.

Since the cells of Sertoli persist and maintain their true characteristics in the cryptorchid testis, it seems reasonable to suppose that they are doing their utmost to preserve the healthy condition of the sperm cells, until such time as the testis is placed in a more favourable environment. If, by the time puberty is reached, the testis is still misplaced, then the cells of Sertoli seem to be unable to prevent atrophic changes in the germ cells. It is 24 Eugenia R. A. Cooper

remarkable, however, to find that the cells of Sertoli are very resistant to the continuance of a malposition, and they persist even in adult life.

Whitehead (40) and Kingsbury(17) described the essential organs in a hermaphrodite horse and pig respectively. They found that the organ in each case was of testicular origin, and in the tubes were cells of Sertoli. In the essential organ in a human hermaphrodite examined in the course of this research, the tubular cells, with a few exceptions, were typical cells of Sertoli. Moore (24) found that in testicular grafts into castrated males the greater number of cells which persisted were cells of Sertoli. Recently Fukui(11) has shown that in “‘heat testis” there is a destruction of the germinal cells, leaving the cells of Sertoli, which with excessive heat also undergo degeneration.

Bissonnette (3) describes the majority of tubular cells in the high-flanker testis in cattle as Sertoli cells.

As the cells of Sertoli are not properly developed until just prior to puberty, it seems natural to assume that they are vitally concerned in the maturation of the germinal cells, which also commences at puberty. Since the former cells are resistant to the continuance of the abnormal position of a retained testis, it is surmised that they endeavour to maintain the healthy condition of the spermatogonia in cryptorchid testes, and are ready to nourish the germ cells, should the testis at any time acquire its normal scrotal position.

6. That the retained gland is able to develop in childhood is seen by the increase in size with advancing age; and although in later childhood the tubules appear relatively less numerous than in the scrotal testis of corresponding age, they are collectively in similar numbers, or even more numerous, than at earlier ages, unless the more unfortunate higher degree of retention prevails.

7. The cells of Leydig are apparently not influenced by the abnormal situation of a retained testis.

In the present research they were seen to be in abeyance in the retained glands of earlier ages, and were present and of typical appearance in these glands at puberty and in the adult. Eccles(9) found the interstitial cells in proportionately large numbers in imperfectly descended testes, and Bissonnette (3) describes these cells as being more numerous in the high-flanker testes of cattle. Moore (23) also remarks on the over-abundance of interstitial cells in retained testes, and states that he was able to reproduce a similar condition in testicular grafts placed in castrated males.

By careful comparison with scrotal testes at similar ages, it is concluded that the interstitial cells were no more numerous in the retained than in the normally situated glands. Since they were only present in the abnormally placed glands examined at puberty and in adult life when, it has been seen, the parenchyma may be in smaller amount than normally, the explanation of their apparent exaggeration appears to be that they become more obvious because the tubules are less numerous and more widely separated. It is of interest to state that a retained testis examined from a human hermaphrodite of thirty-eight years, contained a large number of typical interstitial cells in comparison with the size of the organ and the number of persistent tubes. When compared with a scrotal testis of adult age, these cells collectively did not appear to be excessive as far as could be judged.

8. In no cases of retained testes examined was there anything of an abnormal nature in the structure of the epididymis. The only diversion was that frequently in the retained gland the epididymis and testis were separated by a digital fossa of variable width, but a similar condition was observed also in some scrotal testes, although in smaller percentage.

Bland-Sutton (4), Rawling (33), Turner(38) and Moschcowitz(30) have also drawn attention to the separation of testis and epididymis in imperfectly descended testes. Turner (38) further states that the epididymis, in such an organ, is generally small and ill-developed. In this research, however, the epididymis in the retained testes did not present any abnormal structure.

In conclusion is seems justifiable to advocate the transplantation of the retained testis in all cases during childhood, but especially during the first two or three years, and in those arrested in the lower part of the inguinal canal. Contra-indications for such a procedure are: the persistence of a high abdominal position, when the organ may be removed or left in situ; or the infiltration of the testis with secondary growth of a malignant nature, when removal is the only possible treatment.

Since transplantation of a retained testis into the scrotum is practicable, and may induce spermatogenesis, there should be no hesitation in performing the operation, which will incidentally relieve the individual of his “ unfortunate infirmity.”


==Acknowledgements==

Many thanks are due to A. H. Southam, F.R.C.S., who kindly provided the retained testes for this investigation; to Prof. Stopford of the Anatomy Department of the University of Manchester for allowing this research to be carried out in his department, and for valuable help and criticism; to Prof. Shaw Dunn of the Pathological Department of Manchester University. for permission to obtain material from autopsies at the Manchester Royal Infirmary; to H. Gooding, histologist to the Anatomy Department of the University of Manchester, for preparing the photographs; and to Sir Arthur Keith for welcome advice in the preparation of the manuscript for publication.

==Plates==

EXPLANATION OF PLATES I AND II

. PratE I Fig. 1. Testis of a foetus of 7 months. 4A, interstitial cells. B, spermatogonia. C, undifferentiated cells. x 250 . Fig. 2. On left, retained testis of 14 months (a). On right, scrotal testis of 4 months (8). A, digital fossa. B, epididymis. C, vas aberrans. D, testis. x 250 26 Eugenia R. A. Cooper

Fig, 3. Left, scrotal testis at 11 months (4). x 60. Right, testis retained in inguinal canal, 14 months (B). x 60.

Fig. 4. Retained testis at 24 years. Situated in higher part of inguinal canal. x 60.

Fig. 5. Retained testis at 5 years. Situated at internal abdominal ring. x 60.

Prats IT

Fig. 6 (4) and (B). Retained testis at 6 years, abdominal situation. A, tunica albuginea. B, tubules. C, digital fossa. D, vas aberrans. E, testis. F, epididymis. 68 x 35.

Fig. 7. Scrotal testis at 8} years. x 60.

Fig. 8. Tubules of a testis retained in the inguinal canal, age 13 years. A, spermatogonia. B, undifferentiated cells. x 400.

Fig. 9. Scrotal testis at 12 years, active spermatogenesis. A, spermatogonium with double nucleus. B, spermatocytes; as yet there are no spermatids or spermatozoa. x 250.

Fig. 10. Retained testis, abdominal position, age 15 years. No spermatogenesis. A, group of interstitial cells. x 60.


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