Studies on degenerating sex cells in immature mammals. II. Modes of degeneration in the normal...

STUDIES ON DEGENERATING SEX CELLS I N IMMATURE MAMMALS

11. MODES O F DEGENERATION I N T H E NORMAL DIFFERENTIATION

O F T H E DEFINITIVE GERM CELLS I N T H E MALE ALBINO

RAT FROM AGE TWELVE DAYS T O MATURITY

EZRA ALLEN AND PAUL D. ALTLAND Department of Biology, John B. Stetson University, &Land, Fla., and National

Insti tute of Arthritis and Metabolic Diseases, National Institutes of Health, Public Health Service, Federal Semri ty Agency,

Bethesda, M d .

NINETEEN FIGURES

INTRODUCTION

The mature mammalian seminal epithelium is the most complex of all animal epithelia liistologically, cytologically, and physiologically. Its development is equally complex. Be- fore 1900 the presence of some form of degeneration was noted in apparently normal prepubertal mammals of several species, and came to be regarded as a normal phc- nomenon. The early literature has been ably reviewed by Regaud ( ' lo), Hoven ( '14)' Allen ( '18)' and Hargitt ( '26). Degeneration in all phases of spermatogenesis in the prepubertal rat has been reported by Hoven ('14) and Hargitt ('26)' and for the mouse by Bryson ( '44). The complete degeneration of the so-called "primordial" germ cells has been reported by several observers, notably Hargitt ( '26) and Allen ('49) for the rat at 9-10 days, by Kirkham ('15) for the mouse at 8 days, and by Bryson ('44) for the mouse at 9-10 days after birth. There remains f o r study degeneration in the later stages of development, from the appearance of the spermatocytes to maturity. The modes of degen- generation during this period have been only incompletely

515

516 EZRA ALLEN AND P A U L D. ALTLAND

described. The present paper treats in detail the cytological evidence to be found in albino rats from 1 2 days of age to sexual maturity.

MATERIAL AND METHODS

The testes of 125 normal albino rats were examined cytologically. Of these, 120 came from the Sprape-Dawley colony at the National Institutes of Health; 5, aged 12-16 days, were from slides made from the Wistar Institute colony by Allen (’1s) f o r his study of spermatogenesis. The only difference observed between the two strains was a slight precocity in the lJTistar material. The Sprague-Dawley testes were fixed about one hour in Allen’s B-15 at 38°C. and then transferred to Bouin’s fluid. They were dehydrated by the aniline method, cleared in xylol, embedded in tissuemat, sectioned at 5, 10, or 15 p, and stained in iron hematoxylin. Somc were counter- stained with eosin or orange G. Only the younger testes were entirely sectioned. About 50 sections were mounted from each of the older specimens. The epididymides were f o r the most part completely sectioned. Quantitative estimates of degeneration are fully reported in another paper in this is- sue by Altland and Allen.

TERMINOLOGY

Since the basal cells lining the embryonic testis cord have the potency to give rise to several different types of cell, viz. : “primordial, ” “large” (Allen, ’49), spermatogonia, rete epithelium, and Sertoli cells, we prefer to call them stem cells. We reserve the name spermatogonia for the stem cells which have the potency to differentiate into spermatocytes - definitive germ cells. Allen (’18) used the name A cells for the stem cells and B cells for the spermatogonia, names which have been employed by later writers, e.g. Hargitt (’26) and Roosen-Runge and Giesel ( ’50). The names used in this paper for the modes of degeneration conform to the definitions given in standard medical dictionaries and with common us- age.

DEGENERATION AND TESTIS DEVELOPMENT 517

DESCRIPTIO?: O F THE MODES O F IlEGENERATION

Degeneration in the seminal epithelium of normal postnatal white rats comes to a pause on the 9th day (Allen, ’49) when the “primordial’ ’ cells have practically all been resorbed. During the next 4 days cell multiplication is active and there is little cell degeneration, but on the 14th day a great deal may be present in the more advanced specimens, and it continues in considerable quantity by several modes until the 48th day, when the amount is small, or even nil in some specimens. Throughout this period the degenerate tubules are mingled with normal ones in varying numbers. I n most normal adult white rats sporadic degenerating tubules niay be expected.

In the period between 12 days and maturity 6 principal well differentiated modes of degeneration have been found, all but one of which have been noted in the literature on the rat, other mammals, and man. A few minor modes are not described in this paper because of their rarity. Degenerate sperm are not included in our description of the modes, but are described separately on page 525. The 6 different modes have been named from the stage in their history most commonly seen. They may be classified in two groups, based on different methods of activity. The first group is characterized by loss of cells in compact or disorganized layers, or by the total loss of all germ cells. It includes mode I, exfoliation; mode 11, shedding; and mode 111, necrosis. The second group is characterized by loss through individual cells which occur in small groups, and includes mode IV, pycnosis ; mode V, degenerate leptotene cells not hitherto described ; and mode VI, which contains type 1, abnormal mitotic stem cells, and type 2, abnormal dividing spermatocytes I and 11.

Group I . Loss of cells in layers or by ez t reme Necrosis

Mode I. Exfoliation. I n this mode one or more of the inner layers of normal or degenerating cells dislodge in

518 EZRA ALLEN AND PAUL D. ALTLAND

a compact mass and move centrad (figs. 1-3). The deeper layers remain in position and continue their differentiation normally. Thus only part of the epithelium is lost. This mode is found in both developing and adult animals. I n the rat the history of the mode begins as a narrow slit hetween the inner and outer layers of the epithelium. The kinds of cell set free generally depend on the phase of development present. The exfoliated cells shown in figure 1 were leptotenes. The cells in the next layer were in early prophase. Such tubules were mingled with normal tubules in which the cells lining the lumen were generally zygotenes. Rarely degenerate cells may be exfoliated, in part of a tubule, as: shown in figure 3, while the remainder of the tubule may be normal.

The amount of tissue lost is recognizably large. T'he section of tubule from which figure 1 was made was unusually long, but similar exfoliation extended throughout its length. Very many cells are lost in each tubule so affected. Whether all exfoliated cells are expelled into the epididymis or whether some are resorbed in situ has not been determined, but masses of normal and degenerating cells were found in the epididymis a t all ages older than 15 days.

An exception to the rule that only partial loss of cells occurs by exfoliation was observed in 14-day rats. I n the tubuli recti close to the rete, the stem cclls had developed into a cuboidal or low columnar epithelium, while all the other layers had been set free and were passing en inasse into the rete while still in normal condition. This is the only exception to the rule that we discovered.

Mode 11. Shedding. This mode differs from exfoliation in that normal and degenerating cells of the inner layers separate from each other and move centrad in a disorganized mass (figs. 4-6). At 14 days a few leptotene cells had differentiated and lay isolated in the central plasm (fig. 4). By the age of 16 days more o r less complete layers of leptotene cclls appeared in layers in'which shedding had occurred. This mode becomes most abundant in the aygotene and pachytcne phases and in the spermatid stage. All the phases pass through the

DEGENER,ATION AXD TESTIS DEVELOPMENT 519

same history. The pachytene cells show the process best. After separating, the cells begin to degenerate by vacuolation in the cytoplasm and disorganization in the nucleus. The basal layers of prophase cells are not affected but differentiate normally in. situ. Thus a large part of the epithelium is not lost, although under certain adverse conditions shedding may be the first step in necrosis. I n some longitudinally cut pachytene tubules, shedding appeared in only one level. However, the remainder might be affected later in the same way. Tu- bules which were shedding were ordinarily mingled with normal ones. Exfoliation and shedding might occur close to- gether. For example, only one tubule lay between the two tubules from which figures 1 and 5 were made.

Spermatids shed and degenerate similarly to the pachytene cells. However, in addition to vacuolation a thread-like process may develop from each pole and push out radially between the adjoining cells. MTe may include the discharging of sperm under this mode, as they pass separately into the lumen.

It is no t known whether the degenerate cells due to shedding are finally disposed of by resorption in situ or after passing into the epididymis, but some degenerating zygotencs and spermatids have been found in the epididymis. At any rate the loss by shedding is large, especially among the sper- niatids. Most of these cells apparently degenerate. For example, in a 34-day specirricn an abundance of spermatids was present, but only a small number of sperm appeared.

A few uni- o r multinucleate giant cells were found among the degenerating zygotenes and pachytenes. Judging by the resting state of those similar to figure 9, they were evidently undifferentiated stem cells ivvhich had formed syncytia. These may degenerate im situ (fig. 10). Similar syncytia were noted in postnatal white rats aged 1-9 days (Allen, '49).

Mode 111. Kecrosis. Necrosis differs from the previously described modes by total or nearly total lack of the epithelium. The earliest stage identified in its history is shown in

520 EZRA A L L E N AND P A U L D. ALTLAND

figure 7. The cell elements strongly suggest the relation to necrosis. Quite evidently the central cells are all in. stages of degeneration, while the basal layer is still normal. A later stage was seen in tubules filled with fibrillated plasm, in the center of which was a mass of small unidentifiable cells (fig. 11, A). Along the basement membrane a single row of cells had persisted which were difficult to identify. I n the next stage found, the central cell mass had disappeared, but the tubule was still filled with the mass of fibrillated plasm. In the next to the final stage the plasm had been reduced to a narrow zone inside the basal cells (fig. 11, B). I n the last stage the basal cells were lost; only a thin outer zone of vacuolated plasm lined the basement membrane. The late stages in necrosis are those most commonly seen.

Necrotic tubules were distributed singly and in groups among normal tubules. All the stages described above were found in one such group in a 30-day specimen somewhat underweight. In most specimens we found no necrosis, but in one rat nearly all the tubules were necrotic. I f Sertoli cells are present they may lie among the persistent basal cells, or may be the only cells that survive. Under adverse conditions the loss by this mode may be considerable, but under ordi- nary conditions it is practically nil.

Group I I . Degeneratiom of individual cells Mode IV. Pycnosis. Degenerating cells in this mode are

scattered among normal ones in the epithelium (fig. 12). I n 12-day rats they occurred centrad to the stem cells. They were most abundant at 14 t o 18 days of age. I n the stage of development most commonly seen, as in figure 1.2, the cytoplasm had apparently been resorbed. I n the nucleus a few deeply stained chromatin bodies of different size appeared - only one in the smallest cells. I n all the cells there was some karyolymph present, but without a visible membrane.

The cells in this mode are evidently degenerating leptotenes. (For description of origin see p. 523, 3rd paragraph.) The last visible stage was a tiny, faintly staining homogeneous

DEGENXBATION AND TESTIS DEVELOPMENT 521

body which will be resorbed irz situ (fig. 12, b). Similar tiny bodies have been found in the basal layer of the seminal epithelium of adult rats, the other layers being entirely normal (seen in Ezra Allen’s collection of rat testes). The loss by this mode is relatively not large.

Mode V. Degenerate leptotene cells not previously described. As commonly seen (fig. 14), the cells in this mode possess dense nuclei composed of finely granular chromatin bodies and dense cytoplasm without visible cell membranes. I n the early stage of degeneration the leptotene cells appear to be in abnormal prophase (fig. 14, a), while in the late stages of degeneration the cells stain only faintly (fig. 14, b) and eventually remain only as faintly staining homogeneous bodies (as in fig. 12, b). This stage is resorbed ioz sitze. The loss by this mode is relatively large. It was the most abundant of the individual cell modes and continued present to the age of 40 days.

Mode VI. Abnormal cell division. Two different types of this mode occurred, each at a different stage in the development of the seminal epithelium - one in mitosis of stem cells (fig. 13), the second in the meiotic divisions of spermatocytes I and I1 (fig. 15).

Type 1. I n the first type (fig. 13) the small chromatin bodies are interpreted as abnormal chromosomes OF stem cells in mitosis. This is the condition most cornmonly seen. Tn some cells the bodies were similar to rod-shaped prophase chromosomes ; in others they were clearly in anaphase later than the one marked a in figure 13. The number of chromatin bodies might run as high as 15. The final stage in the history has not been found. This type is rarely met, but has been seen in specimens from 12 to 45 days of age. The cell loss by it is very small.

Type 2. This type was found in anaphase of the first and second spermatocytes. It is shown in spermatocyte I in figure 15. The chromosomes do not behave normally; they as- sume shapes of various sorts which are readily distinguished from those of poorly fixed material. Normal clironiosomes are


shown in figure 16, from the same section as figure 15. The layers of normal cells in these phases are relatively short and narrow in the rat, but during abnormal division the layers in several longitudinally cut tubules appeared long and broad, containing a large number of defective cells. This increase in quantity may be due to acceleration in the rate of division. However, the very numerous spermatids which weire present in a 34-day specimen indicated that an equally large number of normal spermatocytes I and I1 had survived in the preceding days. Spermatocytes in meta- and anaphase began to appear at 24 days, and were rather frequent at 30 days. The further steps in the degeneration and disposition of these degenerate cells have not been seen. Evidently the loss may be large.

Relative loss by the two groups of naodrs The amount of degeneration contributed by exfoliation,

shedding, and necrosis compared with the amount produced by the individual cell modes has not been computed. Thc con- tribution of the cells in the latter group is doubtless considerable. However, wherever present, with the exception of type 2 of mode VI, they never formed more than a small part of the epithelium, so that their loss is small by comparison with the very nunierous cells involved in exfoliation, shedding, and necrosis.

RELATTON O F DEGENl3RATIOPU’ TO EPITFTELIAL LAYER DEVELOPMENT

To understand the relation it is first necessary to review briefly the structure of the adult epithelium and its method of devcloprnent. The adult epithelium lines the testiciilar tubules, and consists of 4 fundamental layers, each a physiologi- cal unit. Each layer is successively different in its cc~ll coni- ponents as the wave of differentiation passes lengthwise in the tubule. The basal layer may consist entirely of undifferentiated stem cells, o r of dividing stem cells o r of B cells, but always has some Sertoli cells. The stem cells arose from


the anlage in the genital ridge, in which they lined the developing testicular cords. They multiply by mitosis, remaining in a single layer as the testis grows. At 10 days after birth they are still in one layer, all similar. Each has a well defined nucleus and nuclear membrane, but the cytoplasm is not differentiated from the plasm of the cord by a visible membrane.

At 1 2 days the layer has become double by mitosis. Appar- ently the cord has not increased in diameter as rapidly as the cells have divided (fig. 17). A very little differentiation was seen, the visible evidence of which was a few B cells which mark the presence of spermatogonia, the first generation of definitive germ cells. These are characterized by the chromatin being in several densely stained somewhat irregu- lar blocks, one or two lying against the nuclear membrane, the cytoplasm not clearly defined (fig. 4, B). These are interpreted as spermatogonia in division; the daughter cells differentiate into the leptotene phase of meiosis.

Besides the B cells there were present in our material, in very small numbers, cells in abnormal mitosis (fig. 13) ; nor- nial and degenerating leptotenes ; and pycnotic cells. The pycnotic cells are interpreted as late stages of degenerating leptotenes. All except the B cells were scattered in the central plasm of the cord. The B cells formed an incomplete layer mingled with the stem cells. Cell division in the stem cells was slight in our specimens, but had increased at 13 days.

By 14 days, in the most advanced individuals, more of the stem cells had formed spermatogonia. In some cords a few leptotenes lay scattered in the central plasm with no definite layer arrangement (figs. 4 and 19). They were still without a visible cytoplasmic membrane, but lay in a vacuole. Great variation in steps of development was found at 14 days. In 10 specimens, for example, 4 showed very few B cells present ; 4 had a few leptotenes in the central plasm ; two showed a ncarly complete layer of leptotenes in a few cords -cut


longitudinally near the surface. Cross sections of cords pre- sented many, few, or no leptotene cells, according to the number in the level cut (fig. 19). No complete layer was found at 14 days.

At 16 days the leptotene layer in many tubules was still incomplete, but in some one or even two layers of leptotenes were found with cells in close order. The latter layers indicated that complete layers of €3 cells had previously been present. The fate of the few leptotenes which had differentiated from the previous generation of B cells is not defi- nitely known. Hargitt ('26) thought that they degenerate.

At 17 days zygotene cells were found shedding into the lumen in some tubules (fig. 18, Z). The layer development at this age comprised complete B layers in some tubules; in other tubules, B cells developing into leptotenes, o r leptotenes differentiating into zygotenes. In one partial layer of zygotenes the cells were enmeshed in a quantity of fibrous plasm, their own cytoplasm also fibrillated. The chromosomes were indistinct. This type of degeneration was distribiited to a slight extent in the specimen. When zygotene cells are shedding, the cytoplasm becomes denser and the chromosomes closely aggregated. Many apparently normal single layers of zygotenes were only partially drawn into the lumen, and would probably have been exfoliated later.

Older stages of developnient. The examples given above suffice to illustrate the chief relations of degeneration to layer development. In the remaining stages the same plan is fol- lowed, viz.:

1. A small number of cells in each new step in differentiation is first produced (figs. 4 and 19) ; many degenerate.

2. The formation of a complete layer follows; many cells degenerate, chiefly by exfoliation and shedding (figs. 1-6).

3. Finally a normally functioning layer of each kind is laid down and degeneration gradually ceases. The epithelium is nearly mature when the normal sperm start shedding into the lumen.

DEGEXERSTION AND TESTIS DEVELOPMENT 525

Sperm development and degeneration The sperm cells do not form a separate layer, but from

very early differentiation mingle with the other epithelial cells. After the heads are well formed they lie in a more or less well defined layer (fig. 8), which moves toward the Sertoli nuclei among the basal cells. Here they seem to complete their development along with that of the tails. Then, still in an irregularly outlined layer, they move to the lumen and are shed. The history of the development of the final layer is like that of the spermatocytes and spermatids - a few sperm first appear, then more and more with increasing age, while many degenerate. The cytological details of the maturing sperm are too complex to be described in this paper.

'The Sertoli cells began to appear at 20 days, fully 10 days before the first signs of sperm differentiation were seen, so that no influence may be ascribed to them on the early steps in sperm formation. Between 27 and 39 days the spermatids had multiplied until most of the seminal tubules resembled the one a t 34 days shown in figure 2. No differentiation into sperm was found a t 30 days. The earliest steps seen were at 34 days, but the presence in a few tubules at that age of a few well formed heads indicated that spermiogenesis had begun somewhere between the ages of 30 and 34 days. The exact age was not learned because of lack of specimens between 30 and 34 days of age. A few spherical, curled, o r enlarged sperm heads were seen among the normal ones at 34 days, and similar ones continued to be present sparingly up to 56 days. There were almost no spermatids, or normal or degenerate sperm in the epididymides until the age of 56 days, when nearly every tube section was filled with apparently normal sperm for the most part. To understand this final developmental period, study is needed of more specimens than were used a t the time this paper was written.

DISCUSSION

1. Historical. Hoven ('14) was the first to make a compre- hensive report on the degeneration of germ cells in normal


prepubertal animals. He stated that degeneration begins about two weeks after birth; at 16 days all the first spermatocytes degenerate and are eliminated into the central canal; also that all first spermatids degenerate and are similarly d!isposed of. Hargitt (’26), after careful study of prepubertal white rats, does not agree with Hoven. He concludes that partial degeneration occurs in all developing cells from 1’2 to 43 days of age. Our observations agree for the most part with Hargitt’s. We have found no later account than his of degeneration in the germinal epithelium of the developing albino rat.

In the prepubertal mouse Bryson (’44) describes degeneration very fully. He lists 4 distinct kinds, viz.: “priinordial germ cell; cytoplasmic vacuolation at the time of lurrten formation ; cellular degeneration of primary spermatocytes and their derivatives in sexually mature mice; severe but local necrosis in the tubules of mice of all ages.” He also described “intensive degeneration of a locally restricted group of cells with no apparent effect on other cells in the surrounding area. The nuclei of this type were shrunken, deeply stained and occupied a position in the tubule which would indicate that many were primary spermatocytes.” They were found in mice of all ages. We have occasionally found similarly located small degenerate cells which are shown in the exfoliated mass in figure 3. Bryson (’44) reports no exfoliation in developing mice. The cells he pictures in his figure 10 he re- gards as asynaptic spermatocytes. They strongly rctsemble our type 1 of mode VI (fig. 13), which we interpret as abnormally dividing stem cells. He concludes that “most of the germ cells of mice under 35 days of age degenerate.” We conclude that our rats had not degenerated to that extent, since they had developed spermatids in large numbers and IZ small number of sperm at 34 days of age. I n fact all the rats we examined froni 34 t o 48 days of age which showed degeneration of various modes possessed a considerable population of sperm in the seminal epithelium.


Sniffen (’50), from a study of prepubertal human testes, reports that at 5 or 6 years of age some germ cells lying centrally are undergoing degeneration. The center of the cord is occupied by degenerating cells, granular debris, a colloid substance, and large binucleated and multinucleated germ cells. From the 12th to the 16th year many spermatogonia die and become necrotic with nuclear “hyperchromatism” and pycnosis. Re concludes that the maturation phenomena in man “seems to be accomplished in successive waves of activity which in the beginning fail the mark, but in time the goal is reached with the formation of mature sperm.” He records no further explanation. 2. Duplication in the modes. All the modes are distinctly

different in the stages commonly referred to in the literature. However, in some modes we found duplication in the early or late part of their histories. For example, the first step in the histories of both types of mode VI is the same - abnormal cell division. The first stage of modes IV and V seems to be abnormal leptotene cells, and their final visible stage is a tiny pale homogeneous body (figs. 12 and 14, b). There is some evidence that necrosis may originate by exfoliation o r by shedding. For example, when adult rats were subjected to severe hypoxia (Dalton et al., ’45; Altland, ’49) large masses of immature germ cells apparently originating by exfoliation were found in the lumen of the seminiferous tubules and in the epididymis. Seminal tubules in the adult rats (Dalton et al., ’45) became necrotic. Extensive shedding was found in the seminal tubules of LZ rat somewhat underweight and described under necrosis, page 520. There is no other duplication in the histories of exfoliation, shedding, and necrosis unless it may be learned that all the degenerate cells of these modes pass into the epididymis.

3. Factors of degeneration. The plan of seminal epithelial differentiation described above indicates that degeneration may be regarded as a function of development. Such an inter- pretation is in line with the fact that degeneration is an essential factor in the development of many animal organs.


It is one of the many factors in the whole process of clevelop- ment, most of which are not yet understood. The factor of degeneration is itself complex and is not fully understood. However, in our study certain conditions have been observed which appear to have definite influence on testicular degeneration. These include crowding of cells, insufficient supply of local energy, genetic constitution, and adverse somatic and external conditions.

Crowding of cells may be classed with the fundamental developmental processes. During growth of the testis, physical relationships play a large part. The seminal tubales increase in length and in diameter and the cells incrsease in number and size. For example, the single layer of stem cells a t 10 days has doubled at 12 days, and many cells have elon- gated radially (fig. 17), as if the available space was too short. Later, when the larger B cells become more numerous, the picture is one of a dense mixture of A and B cells. The B cells seem to struggle f o r the outer position, where they ultimately arrive (fig. 4). It is during this period and a little later that mode IV, pycnosis, and type 1 of mode VI, abnormal mitosis, appear in greatest number.

The larger leptotene cells next appear and perha.ps because of crowding do not differentiate synchronously and form a complete layer (fig. 4). As a result the more precocious ones are pushed into the yielding plasm. There, out of con- tact with the inductive influence of their sisters and the A and B cells, they may not survive. Some of them pass into the zygotene phase (fig. 18) and scatter in the central plasm as isolated cells. If B cells are not differentiated into a well- formed layer, it is doubtful if a complete layer of leptotenes may be formed. Some of the scattered leptotene cells apparently degenerate irz sitzc, since they are not found in the rete or epididymis a t 14 days (Hargitt, '26). We have observed similar conditions at 15 days also. However, these losses do not exceed the margin of safety because complete layers of leptotenes are formed later at 16 days. In the later stages of differentiation exfoliation and shedding as well as

DEGENEEATION AND TESTIS DEVELOPMERT 529

some abnormal cell division may be due to crowding. I n no. 102, a 27-day rat which was somewhat underweight, exfoliation and shedding were widespread ; many tubules were filled solid with either normally developing or degenerating cells.

I n all specimens the great amount of shedding among the zygotene and pachytene cells may be caused in part by crowd- iiig due to the increase in size of these cells. One is im- pressed by this enlargement when tracing the growth from the earliest differentiation of the zygotene to the fully developed pachytenes.

The testes are in the inguinal canals during the ages of 23 and 28 days when spermatocyte differentiation is active (Hargitt, ’26). This environment may be favorable for crowding.

Factors not fundamentally developmental may also pro- duce degeneration. Insufficient local energy supply may be such a factor. Cell division is normally more active in prepubertal mammals than in adults. During the latter part of meiosis the complex chromosomal activities may demand increased energy. Perhaps the degenerating cells may supply this need at this period as well as a t other times and thus become expendable, like some germ cells which become nurse cells in certain invertebrates, as in Moina ?nacrocopn (Allen and Banta, ’29). Bryson ( ’44) concludes that nuclear degeneration of spermatocytes “is a morphological manifesta- tion of the inability of cells in immature animals to go through a complete spermatogenetic cycle. ” He does not analyze the term “inability. ” Undoubtedly insufficient energy, from what- ever source, is one factor. We do not agree that all the developing germ cells fail. The opposite is clearly illustrated by the large number of spermatids that develop synchronously with abundant degenerating first and second spermatocytes.

Some of the deleterious effects of hypoxia have already been described on page 527. Hypoxia also results in f a ih re of sperm differentiation (Altland and Allen, ’52). Other ef-

530 EZRA ALLEN AND P A U L D. ALTLAND

fects of hypoxia are also discussed in that paper. It is possible that unequal blood supply rnay account for more degeneration in some tubules than in others nearby. Often increased degeneration is found in several tubules close to- gether, as though under common influence, while most of the more distant tissue is normal. It is well known that hormonal abnormalities and insufficient food and unbalanced metabo- lism due to various somatic conditions result in increased degeneration. The great variation in quantity of degeneration among rats under the same environmental conditions is not yet understood. One factor may be genetic constitution. An illustration is found in Bryson ('44), wlio concludes from his study of heterozygous mice that somatic influence, not chromosomal incompatibility, accounted for more than 50% of abnormal sperm in his hetcrozygote tot1.

In their reaction to disease and other adverse conditions, both somatic and external, the developing cells show great sensitivity. The excess of exfoliation, shedding, and necrosis in rats somewhat underweight has already been described on pages 520, and 529. The higlier temperature of undescended testes seems to be the cause of failure of seminal differentiation. Similar results were obtained by Moore ( '26) and Nel- son ('51) by replacement of testes for some time in the body cavity of rats. They reported also exfoliation, giant cells, and nearly total degeneration. It is noteworthy that no different modes of degeneration other than those reported above have been described in rats subjected to abnormal conditions.

SUMJIARP A S D C'OXCLTXIONS

1. Cytological examination of the testes of 125 normal postnatal albino rats, aged 12 days to maturity, revealed 6 major modes of degeneration in two groups: the first characterized by the loss of entire layers of cells, the second by individual cell degeneration. Group 1 includes three modes : exfoliation, shedding, and necrosis; group 2, three modes: pycnosis, de-

DEGENERATION AKD TESTIS DEVELOPMEST 531

generate leptotene cells not previously described, and two types of abnormal cell division, one associated with stem cells, the second with spermatocytes I and 11. The history of the modes is described.

2. Great individual variation was found in the variety and quantity of degeneration: modes I and I1 provided the greatest loss of cells; modes I, 11, and V were the most widely distributed.

3. Degeneration reached its peak in the zygotene, pachytene, and spermatid stages. 4. Modes I, 11, and type 2 of mode VI are regarded as

normal processes under the influence of the fundamental forces of development; modes 111, IV, V, and type 1 of mode VI as chiefly due to adverse internal and external conditions.

5. Degeneration is intimately associated with the differentiation of the seminal epithelium. The plan of development is as follows: at 1 2 days spermatogonia begin to be differentiated from stem cells; a t 14 days an incomplete layer of cells in the leptotene phase is laid down which is accompanied by degeneration. But, a sufficient margin of safety prevails to provide a source of normal cells for a complete layer later. The same method follows with the successive phases of meiosis and with spermiogenesis until the normal epithelium is complete. The function of degeneration appears to be at least two-fold: one to provide sufficient space for the rapidly differentiating cells, the other to help supply needed energy. The seminal epithelium is nearly complete at 48 days, the age when some male rats have proved capable of fertiliza- tion. Fully mature spermatozoa are abundant at 56 to 60 days.

6. The relations of constant physical factors, such as cell crowding, and the possible influence of such variable factors as an insufficient energy supply, constitutional genetic anomalies, and somatic and environmental abnormalities to germ cell degeneration and differentiation are discussed.

532 EZRA ALLEN A N D P A U L D. ALTLAND

LITERATURE CITED

ALLEN, EZRA 1918 Studies on cell division in the albino rat. 111. Spermato- genesis: thr origin of the first spermatocptes and the organization of the chromosomes, including the accessory. J. Morph. and Phpsiol., 32 :

1949 Studies on degenerating sex cells in immature mammals. I. An analysis of degeneration in primordial and large germ cells in male albino rats aged 1-9 days. J. Morph., 85: 405-422.

Growth and maturation of the partheno- genetic and sexual eggs of Moina macrocopa. J. Morph. and Physiol., 48: 123-151.

1949 Effects of discontinuous exposure t o 25,000 feet simulated altitude on growth and reproduction of the albino rat. J. Exp. Zool., 110: 1-18.

Studies on degenerating sex cells i n immature mammals. 111. The influence of hypoxia on the degeneration of primordial and differentiating definitive germ cells of male rats. J. Morph., 91: 541.

BENDA, C. 1887 Untersuchungen uber den Bau des fnnktionierenden Samen- kanalchens einiger Saugethiere und Folgerungen fur die Spermato- gcnese dieser Wirbelthierklasse. Arch. f . mikr. Anat., 90: 49-110.

Spermatogencsis and fertility in Mus musculus a s affected by factors a t the T locus. J. Morph., 7 4 : 131-187.

1945 Organ changes in rats exposed repeatedly to lowered oxygen tension with reduced barometric pressure. J. Nat ’1. Cancer Institute, 6 : 161-185.

The formation of the sex glands and germ cells of mammals. 11. The history of the male germ cells in the albino rat. J. Morph. and Physiol., 42: 253-305.

HOVEN, H. 1914 Histog6nPse du testirule des mammifhres. Anat. Am., 4 7 : 90-109.

KIRKIIAM, W. B. 1915 The germ cell cycle in the mouse. Anat. Rec., 10: 217- 219.

MOORE, C. R. 1926 The biology of the mammalian testis and scrotum. Quart. Rev. of Riol., 1: 4-50.

NELSON, W. 0. 1951 Mammalian spermatogenesis : Effect of experimental cryptoorchidism in the rat and nondescent of the testis in man. Re- cent Progress in Hormone Research, 6: 29-62.

REGAUD, C. L. Etudes sur la structure des tubes seminifhres et sur la spermatogenese des mammifhres. Arch. d’anat. Micr., 11 : 291-431.

ROOSEN-RUNGE, E. c., AND L. 0. GIESEL, JR. 1950 Quantitative studies on spermatogenesis in the albino rat. Am. J. Anat., 87 : 1-30.

SNIFFEN, R. C. 1950 The Testis. I . The normal testis. Arch. Path., 50: 259- 284.

133-186.

ALLEN, EZRA, AND A. M. BANTA 1929

ALTLAND, P. D.

ALTLAND, P. D., AND EZRA ALLEN 1952

BRYSON, VERNON

DALTON, A. J., B. 3’. JONES, V. B. PFTERS AND E. R. MITCHELL

1944

HARGITT, G. T. 1926

1910

PTA TES

BXPLANI~TION O F PLATES

All figures a r e pliotonijcrogmplis iiiude at the iiiagiiificaticms s ta ted and not rcduccd in pulJ1ic:rtion. The lemes varied iritli the magnification desired. They inclniled R Eausch and Lomh 4-nirii and 1.8-nini oil immersion, and a Z(*iss 1.5-mm oil iinmersion objective; Zeiss 0 pro.jrction und Lcitz plaiiar 5 x oeiilars.

The sections were stained with iron hematoxylin.

PLATK 1

CXPLANATION O F FIClIRI%,S

1 Exfoliated leptotencs di5lodgrcl f r o m n 1;i)er of ,! oiingci leptotcnes n n d h s a l stem cells in :I 29-day tubule.

2 Exfoliated sperixltids (light) and dcgrnerating lcptotenes (dark) SUP

rounded by a wide layer of spermatids; the next I:i,wr, well :idTanced leptotcnes; basal cells, rcstiiig stcin cells. Agr, 34 days. X 260.

3 Exfoliatrd lrptoteiies, the c1:irk ones dcyperating, dislodged from leptotenes; basal cells, resting stem cells. Age, 27 days.

4 A 14-day cold with piecocious normal Icptotenes in the central plasm. R rells have crowded the stem, or A cells, and some 13 cells centrad; a fern hnsnl cells in mitosis. X 600.

Pachytene cells shedding f rom a Iaqer of lrptotenes; outer layer, resting stem cells; photograph of the second tuhiile f rom the tubule shown in figurc 1. Age, 29 days. X 250.

6 A 27-day tubule. The central diplotene cells hare becu shed. The next layer is composed of leptotene crlls, the basal layer of stem cells.

7 A 29-day tubulr. The outer laycr is composed of noriiial R cells iii two ro\\s. The central cells ale :ill degenerate. The one marked a has reached tlic homogencous condition dcserihed under pycnosis :ind marked b in figure 12. Thc central plusni is seatteicd iii siiiall ninsses. T t is iegardrd as :in early stage iii

nccrosis. A 3R-d:iy tuhulc u i th a few Hell-formed sperm heads; no abnormal ones

in focus, though some appear abi~o~i iral beenuse of the focus. Thcy lie among diplotciie cells wliiclr :lie surrounded hv re ly earl) lrptoteiics, which obscure the basal stem cells. X 400.

X 250.

X 250.

5

X 250.

8

534

L, E G K S E I< .\TI OX AS I1 TL: STl S D l? 1‘ E LOP31 K N T ELI<.\ ALLLS AZID I’ALL U. ALTLAND

PLATE 1

535

PLATE 2

EXPL4NATIOA' OF FIGLTRICS

9 A giant cell among pachytene cells, interpreted ns a syncytiuin of stein cells. The lone small nuclrus is one of the p:icbytencs. Age 29 &ips. X 1200.

10 A similar cell, \\hie11 shows clcgeneration very clearly. It lics aiiiong zpgotrne cells. Agc, 34 days. X 700.

11 Two adjarent necrotic tubules fiom a groiip of similar ones in a. speciineii somewhat undeiweiglit. Tlie st:igc niarlted A is earliei th:in the one marked B, \+liich is :ilmost completely necrotic. The cells in th r center of A are not iecognizable; the suironnding p1:rsin is filirillated. The cptoplasni in R has lost the fihrillated consistcncp. Age, 30 dn js . X 400.

A typical picturc of mock IV, pJciinsis. The last visible stage of degeii- eration is niaiked b. Age, 14 days. X 1000.

Stem cells in abnormal mitosis, typv 1 of mode VI. The cell niniked a is in snaphase. Not all the cells in tlic ~ i l i ~ ~ l e gioup are in focus. Age, 14 days. X 1200.

A typical group of mode lr. Roinetimes tlicica arc tizo or three more of the large cells. The nnnir~oiis gr:mulcs of the nucleur (lo iiot sliom clcailp; thc cytoplasm is typical. The coll niaiked a is :in abnormal lrptotene. the first stage i n the history of thc mock; the cells marked b are almost a t the other end r t t ' the histor?, approacliing the sinall, Iioniogeneons cells markrd b in figure 12. -\ge, 24 days. X 1200.

1.7 Degenciate sl)eniatocytcl 1 in nbnoininl division; type 2 nf niode VI. Age,

16 Normal speimntoeptc I in nlet:~ph:ise, from sainc microscopic section a s

1 2

13

14

34 d;qs. x 1200.

figure 15. Agc, 34 days. X 1200.

PLATE 2

5 3 i

PLA4TE 3

EXPLAYATION O F FIGURES

1 7

18

Section of a 12-day cold. Note the double row of cells many of which arc eloiignted radially as if squeczed h y crowding.

A 16-day tuhiile nitli t\+o zygotene rrlls ( Z ) , the ceiifral onc a1rr:tdy shrd, sho\+iiig the XY complcx; the other still niriong tlic outer cells. In seine tubules tlirrr are nuiiicrous cciitrnlly located zygotene rells.

Hrction of n l l o l l i i n~ 14-day testis shORiilg the ra r j ing stages of 1cptotrrie differentiation. The cord maiked ‘ ‘ a ” lias degenciate rentwl cell.;, probably leptoteiies; “ b ” slioizs ii cord cut sngitt:illy just inside the basal layer, niid 1i:iving 8x1 exceptional number of leptotrnc cells; “ c , ” :I cold n i th a. fcw widely spaced leptoteries; “d ,” a rord with no leptotenr rells, some of the l)as:~l cells iii division. The other cords pieseiit similar derelopmental x i i i iatioirs. x 300.

x 1200.

X 1200. 19

538

Studies on degenerating sex cells in immature mammals. II. Modes of degeneration in the normal...

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