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Ultrastructural and cytochemical changes in spermatogonia and Sertoli cells of whole-body irradiated mice.

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Ultrastructural and Cytochemical Changes in
Spermatogonia and Sertoli Cells of
Whole-body Irradiated Mice
J. HUGON
AND M. BORGERS
Laboratoire de Microscopie Electronique, Dkpartement de Radiobiologie,
Centre $Etude de l'Energie NucUaire, MOL, Belgique
ABSTRACT
Adult male mice of CBA/C57 B1 strain received a whole-body x-irradiation of 400 r. The ultrastructural and cytochemical changes of the spermatogonia and of
the Sertoli cells were studied throughout the first 48 hours after this irradiation.
Tissues were fixed in osmium tetroxide or glutaraldehyde. The acid phosphatase reaction was carried out using a slightly modsed Gomori medium. The first changes
observed in spermatogonia were: dilatations of the smooth endoplasmic reticulum,
moderate enlargement of the mitochondria and swelling of the nuclear membranes.
In a later stage, the injured spermatogonia were phagocytized by the adjoining Sertoli
cells, where the complete involution took place. The study of the acid phosphatase
localization showed that the numerous dense bodies, appearing in the Sertoli cells in
the first hours after irradiation, were heavily loaded with reaction product. They
seemed to take an important part in the resorption of the phagocytized spermatogonia.
These results were compared with findings of other authors.
The radiosensitivity of germ cells was
described for the first time in 1903 by
Albers-Schonberg. In 1906, Regaud and
Blanc demonstrated that spermatogonia
were the most sensitive of the germ cells.
The progressive loss of sperm cells after
exposure to irradiation was ascribed by
Eschenbrenner and Miller ('50) and Shaver
('53) to an inhibition of mitosis. Oakberg
in 1955 estabIished that the sterility was
due to a true degeneration of spermatogonia with a maximum at 12 hours postirradiation. At the ultrastructural level,
Nebel described in 1958 the dead spermatogonia of the mouse, a few days after
1,000 r. In his study on rat testes irradiated with 10,000 r and 50,000 r, Lacy ('62)
found, eight days after irradiation, many
irregular bodies in the Sertoli cells and concluded that they were the remnants of irradiated spermatogonia.
The purpose of this paper is to present
our findings on the early ultrastructural
changes and on the localization of acid
phosphatase in the spermatogonia and
Sertoli cells of mice, after a single dose of
400 r of x-rays.'
MATERIAL AND METHODS
Adult male mice of strain CBA/C57 B1
received 400 r of x-rays as whole-body irANAT. REC.,155: 15-32.
radiation; the conditions of irradiation
were: 300 kV, 20 mA, 100 r/min. The
animals were sacrificed 1, 2, 4, 8, 16, 24
and 48 hours after irradiation. Control
mice were sacrificed at the same times.
Testicular tissue was fixed immediately in
OsO, 1% for one hour, dehydrated in a
graded series of alcohol and embedded in
Epon. For the cytochemical study, the
fixation was carried out in glutaraldehyde
6.25% for three hours (Sabatini, Bensch
and Barnett, '63). The small blocks were
stored overnight in 0.1 M cacodylate buffer
(pH 7.4), containing 0.22 M sucrose. Frozen sections 50 p thick were incubated in a
modified Gomori solution (Hugon and
Borgers, '65) for 30 minutes, postfixed in
Os04 2% for 30 minutes and embedded in
Epon. Control sections were incubated
without substrate or in a complete medium
containing NaF 0.01 M. Thin sections
were stained with uranyl acetate followed
by lead citrate and examined in a Philips
EM 200 microscope.
1This work was done thanks to the contract
and thanks to grants
from the Fonds de la Recherche Scientifique Fondamentale Collective.
2 A short abstract of part of this work was exposed
at the Third European Regional Conference on Electron Microscopy, Prague, 1964, p. 257.
Euratom/C.E.N., 053-64-3 BIOB
15
16
J. HUGON AND M. BORGERS
RESULTS
A. Control mice
Spematogonia are seen as round shaped
cells situated between the Sertoli cells
along the basal membrane of the tubule
(fig. 1 ) ; their Golgi apparatus and endoplasmic reticulum are poorly developed
with few tubules and vesicles; many microtubules are present in the cytoplasm
around large nuclei; dense bodies are
rarely found. According to the description
of Andre ('62), the spermatogonia of type
A and €3 can be distinguished by the internal structure of their mitochondria : the
former have straight dense cristae (fig. 2 ) ,
the latter have curved cristae with a certain degree of intracristal dilatation (fig.
3). In the B spermatogonia, the nuclear
chromatin is coarser.
Sertoli cells have large irregular mitochondria with tubular cristae; the smooth
endoplasmic reticulum is well developed
with many vesicles; large lipid droplets
are frequent at the base of the cell; their
appearance may be connected with the
stage of the germ cell maturation; 4 to 7
dense bodies of different shapes are observed per cell section. The nuclei are
large and indented with very prominent nucleoli. Fine layers of cytoplasm surround
spermatocytes and spermatids; where two
Sertoli cells meet, their plasma membrane
show a modification with regular bands of
condensed electron opaque material, approximately 400 A wide, on the internal
side of the opposing double membrane;
under this densified structure, dilated profiles of smooth endoplasmic reticulum are
elongated (figs. 4, 5).
In agreement with findings in the rat
testis (Tice and Barnett, '63) the localization of acid phosphatase is different in the
gonia, the spermatocytes, the spermatids
and the Sertoli cells. In spermatogonia,
few vesicles close to the Golgi apparatus
and the very rare dense bodies show a
lead precipitate. In late spermatocytes and
spermatids, where the Golgi apparatus has
a special curved shape with concentric
tubules and vesicles, the phosphatase reaction is always very pronounced in the
more internal cisternae (fig. 6). In the
Sertoli cells, the acid phosphatase activity
is not observed in the Golgi vesicles and is
only situated in structures related to dense
bodies (fig. 7 ) ; circular dense vacuoles
(fig. S), irregular bodies with vesiculated
inner structures (figs. 9, lo), myelinic figures, and complex bodies with double limiting membranes. The latter are probably
the residual bodies described by Lacy ('62).
These structures are frequently located
very close to lipid areas.
B . Irradiated mice
Oakberg ( ' 5 5 ) has demonstrated that
the irradiated spermatogonia begin to degenerate when they enter the prophase
period; there is, of course, a wave of cytolysis with a maximum between 8 and 12
hours post-irradiation. Also, in the same
tubule, the spermatogonia present different lesions according to the period of the
mitotic cycle during which irradiation is
given and an accurate timing of the alterations is difficult to establish.
The first changes observed in some spermatogonia as early as one hour after irradiation are: dilatations of the smooth
endoplasmic reticulum forming many vesicles, moderate enlargement of the mitochondria, disturbance of the straight arrangement of their cristae in the A gonia
(fig. 12), and swelling of the nuclear
membranes.
In a later stage after irradiation, the
changes concern the plasma membrane between gonia and Sertoli cells: first, the
plasma membrane of the gonia presents
large disruptions (figs. 13, 14); afterwards,
the surrounding Sertoli membrane disappears and the spennatogonia are engulfed
in the Sertoli cytoplasm. From the fourth
hour after irradiation, the number of these
phagocytized spermatogonia increases to
include, at the end of the first day, the
great majority of gonia. The absorbed cells
disintegrate quickly (fig. 15) : the nuclei
become picnotic and the cytoplasmic structures degenerate into myelinic figures (fig.
19); the remnants of these phagosomes
have the appearance of the residual bodies
observed in the normal Sertoli cells.
Some spermatogonia, which were probably in prophase just before irradiation,
are able to finish their nuclear division but
fail to complete the cytodiaeresis and give
rise to double nucleated giant cells; there
ULTRASTRUCTURE O F JERADIATED TESTES
is no evidence of phagocytosis of these
cells by the Sertoli cells.
A few young spermatocytes have an
identical evolution and are engulfed by the
Sertoli cells or form multinucleated cells.
The ultrastructure of the Sertoli cells is
modified during this process; the smooth
endoplasmic reticulum proliferates greatly
and many distended vacuoles surround the
spermatogonia; dense bodies become very
numerous forming a layer close LO the
spermatogonial membrane (fig. 18) ; after
the ingestion of the gonia they remain
around the disorganized cells.
The cytochemical study shows that,
after irradiation, there is no modification
in the localization or in the intensity of
the acid phosphatase reaction in spermatogonia, in spermatocytes or in spermatids.
Once the germ cells are absorbed by the
Sertoli cells, however, they seem completely devoid of any lead phosphate deposit. In the Sertoli cells, the numerous
dense bodies are heavily loaded with lead
precipitate (figs. 16, 17). They remain
very close to the remnants of the germ
cells but are not encountered in the necrotic area's as long as no membrane surrounds these areas. Nevertheless at a
later stage, i.e., 48 hours after irradiation,
these phagosomal remnants with limiting
membrane present a diffuse positive reaction (fig. 20).
DISCUSSION
The ultrastructural aspect of testis cells
of normal mouse (Gardner and Holyoke,
'64) seems almost identical to that of the
rat and other rodents (Challice, '53; Burgos
and Fawcett, '55; Zebrun and Mollenhauer,
'60; Prokof'yeva-Bel'govskaya and Chan
Chun He, '61). The complicated membrane relationship between two Sertoli cells
is very similar to the structure described
in the rat testis by Brokelmann ('63).
In relation to the cytochemical observations, it may be noted that the special localization of acid phosphatase in the Golgi
cisternae of spermatids was mentioned by
Novikoff and Goldfischer ('61). In Sertoli
cells, all the morphological types of lysosomes were observed, ranging from the
simple dense vacuoles to the complex bodies with myelinic figures and irregular internal vesicles. It may be that the latter
17
represent a final stage of digestion of the
phagosomes (residual bodies of Lacy ('62)).
From our irradiated experiments, two
main observations have been made : the
morphological process of the disappearance
of gonia in the Sertoli cells; the increasing number of lysosomes in the Sertoli
cells, a short time after irradiation.
These results have to be compared with
the findings of other authors.
Oakberg ('55) definitely established that
irradiation lulls the spermatogonia and
does not block mitosis. It is likely that the
necrotic spermatogonia that he counted
after irradiation were already engulfed
spermatogonia with broken-down membranes. The fast disappearance of these
necrotic cells explains that histological
preparations, made a few days after irradiation, do not show many dead spermatogonia. The phagosomal remnants, indeed,
are not easy to observe with the light
microscope (Casarett and Casarett, '57;
Eschenbrenner and Miller, '50; Eschenbrenner, Miller and Lorenz, '48).
The phagocytosis of irradiated gonia by
Sertoli cells has been described by Nebel
('59) and by Lacy ('62). These authors,
however, made their observations one week
after irradiation and, at this moment, it is
too late to notice the beginning of the
process. Nevertheless the start of this
evolution seems very important. The
ultrastructural lesions of spermatogonia
being engulfed are not too severe and include the dilatation of smooth endoplasmic
reticulum and the slight enlargement of
the mitochondria.
The quick recovery of these changes
have been observed in many other cells
(Hugon, Maisin and Borgers, '65b) even
after x-rays (Hradil, '63). In the germinal
epithelium, however, Sertoli cells immediately disrupt the plasma membranes of
the gonia and the true degeneration with
picnotic nuclei and rupture of the mitochondria takes place when the gonia are
directly in contact with the cytoplasm of
the Sertoli cell. These observations suggest that injured spermatogonia release a
substance which induces the phagocytic
capacity of the Sertoli cell in a similar
manner as dead leucocytes stimulate white
blood cells (Bessis, '64). Another possibility is that the spermatogonial membrane
18
J. HUGON AND M. BORGERS
loses a protective layer which normally
isolates the spermatogonia from the Sertoli
cells.
The increase of cytochemically detectable acid phosphatase has not the same
morphological aspect as in the stem cells
of duodenum (Hugon, Maisin and Borgers,
'65a; Hugon and Borgers, '65). The numerous small vesicles loaded with the enzyme, apparently originating in the Golgi
zone or formed in the ergastoplasm (protolysosomes of Gordon, Miller and Bensch,
'65; or virgin lysosomes of Moe, Rostgaard
and Behnke, '65) are not found in the
Sertoli cells. Only numerous dense bodies
heavily loaded with lead phosphate are
prominent in these cells. These lysosomes
are large, occasionally vacuolated, like
hepatic lysosomes (Essner and Novikoff,
'61 ; Trump and Ericsson, '64). Lead phosphate precipitate is not observed in the
necrotic area surrounded by lysosomes.
Afterwards, it may happen that one or two
lysosomes fuse with the phagosomal remnants.
The high phagocytic capacity of the
Sertoli cell after x-rays may throw some
light on the observations of Berliner and
Ellis ('65) who showed that after inadiating rat testes with 1,500 r, there was a
pronounced increase of 1 7-a-20sr-dihydroxyprogesterone. Lacy ('62) demonstrated in
Sertoli cells a secretion of hormonal nature
probably induced by the residual bodies.
He quoted non published results of Stempfel who observed a secretion of dihydroxyprogesterone by Sertoli tumor. The great
number of phagocytized spermatogonia
may explain in part the results of Berliner
and Ellis.
It may be noted that young spermatogonia seem to react differently, since Sapsford ('62) reported that in x-irradiated
testes of young mice, the gonocytes exhibit
an excessive increase in size and apparently do not die.
LITERATURE CITED
Albers-Schonberg, H. 1903 Ober eine bisher
unbekannte Wirkung der Rontgenstrahlen auf
den Organismus der Tiere. Munch. Med.
Wochenschr., 50: 1859-1860.
Andr4, J. 1962 Contribution la connaissance
du chondriome. J. Ultrastruct. Res., Suppl. 3
1-185.
Berliner, D. L., and L. C. Ellis 1965 The effects of ionizing radiations on endocrine Cells.
IV. Increased production of 17-a-2O-a-dihydroxyprogesterone in rat testes after irradiation. Radiat. Res., 24: 368-373.
Bessis, M. 1964 Studies on cell agony and
death: a n attempt a t classification in cellular
injury. Ciba Foundation Symposium, J. & A.
Churchill, Ltd., London, pp. 287-316.
Brokelmann, J. 1963 Fine structure of germ
cells and Sertoli cells during the cycle of the
seminiferous epithelium in the rat. Z. Zellforsch., 59: 820-850.
Burgos, M. H., and D. W. Fawcett 1955 Studies on the fine structure of the mammalian
testis. J. Biophys. Biochem. Cytol., I: 287-300.
Casarett, A. P., and G. H. Casarett 1957 Histologic investigation of mechanisms of X-ray effects on spermatogenesis i n the Rat. University
of Rochester Atomic Energy Project. UR-496/7.
Challice, C. E. 1953 Electron microscopy studies of spermatogenesis in some rodents. J. Roy.
Microsc. SOC., 73: 115-127.
Clerg, E., and E. McMillan 1965 The utpake
of vital dyes and particulate matter by the
Sertoli cells of the rat testis. J. Anat. (London), 99: 219-229.
Eschenbrenner, A. B., and E. Miller 1950 Effect of roentgen rays on the testis. Quantitative histological analysis following whole-body
exposure of mice. Arch. Pathol., 50: 736-749.
Eschenbrenner, A. B., E. Miller and E. Lorenz
1948 Quantitative histologic analysis of the
effect of chronic whole-body irradiation with
Y rays on the suermatoeenic elements and the
intektitial tissue of thetestes of mice. J. Natl.
Cancer Inst., 9: 133-147.
Essner, F., and A. Novikoff 1961 Localization
of acid phosphatase activity in hepatic lysosomes by means of electron microscopy. J.
Biophys. Biochem. Cytol., 9: 773-784.
Gardner, P., and E. Holyoke 1964 Fine structure of the seminiferous tubule of swiss mouse.
I. The limitine membrane, Sertoli cell, spermatogonia a d spermatocytes. Anat. Rec.,
150: 391-404.
Gordon, G., L. Miller and M. Bensch 1965
Studies on the intracellular digestive process
in mammalian tissue culture cells. J. Cell Biol.,
25: 41-55.
Hradil, I. 1963 Some changes in the cytoplasm
of Liver Cells after X-ray Irradiation, Electron
Microscopic study. Folia Biologica (Praha), 9:
287-291.
Hugon, J., and M. Borgers 1965 Etude morphobgique e t cytochimique des cytoIysomes de
la crypte duodhale de souris irradike par
rayons X. J. Microsc., 4: 643-656.
Hugon, J., J. R. Maisin and M. Borgers 1965a
Changes in ultrastructure of duodenal crypt in
X-irradiated mice. Radiat. Res., 25: 489-502.
1965b Modifications ultrastructurales
aprhs radioprotection. Symposium sur les Radioprotecteurs, Lihge, juin (to be published).
Lacy, D. 1960 Light and electron microscopy
and its use in the study of factors influencing
spermatogenesis in the rat. J. Roy. Microsc.
SOC.,79: 209-225.
1962 Testis structure and function.
Brit. Med. Bull., 18: 205-208.
ULTRASTRUCTURE OF IRRADIATED TESTES
Moe, H., J. Rostgaard and 0. Behnke 1965 On
the morphology and origin of virgin lysosomes
in the intestinal epithelium of the rat. J.
Ultrastr. Res., 12: 396403.
Nebel, B. R, 1959 Fine structure of chromosomes in man and other metazoa and testicular
recovery from X-rays in mammals. In: Progress in Nuclear Energy. Biological Sciences,
Pergamon Press, London, pp. 404410.
Novikoff, A. B., and S. Goldfischer 1961 Nucleosidediphosphatase activity in the Golgi apparatus and its usefulness for cytological studies. Proc. Natl. Acad. Sci., 47: 80S8-810.
Oakberg, E. F. 1955a Sensitivity and time of
degeneration of spermatogonic cells irradiated
in various stages of maturation in the mouse.
Radiat. Res,., 2: 369-391.
1955b Degeneration of spermatogonia
of the mouse following exposure to x-rays, and
stages in the mitotic cycle at which cell death
occurs. J. Morph., 97: 39-54.
Prokof'yeva-Bel'govskaya, A. A., and Chan Chun
He 1961 Electron microscopic investigation
of spermatogenesis in the mouse. Biophysics,
6: 764-776.
Regaud, C., and J. Blanc 1906 Action des
rayons X sur les diverses gbnhrations de la
19
lignbe spermatique. Extr&me sensibilitb des
spermatogonies a ces rayons. C. R. SOC.Biol.,
61: 163-165.
Sabatini, D. D., K. G. Bensch and A. J. Barnett
1963 Cytochemistry and electron microscopy:
the preservation of cellular ultrastructure and
enzymatic activity by aldehyde fixation. J.
Cell Biol., 17: 19-58.
Sapsford, C. S. 1962 Changes in the cells of
the sex cords and seminiferous tubules during
the development of the testis of the rat and
mouse. Austr. J. Zool., 10: 178-192.
Shaver, S. L. 1953 X-irradiation injury and repair in the germinal epithelium of male rats.
Am. J. Anat., 92: 391-432.
Tice, L. W.,and R. J. Barnett 1963 The fine
structural localization of some testicular phosphatases. Anat. Rec., 147: 43-64.
Trump, B., and J. Ericsson 1964 Electron microscopic observations on the localization of
acid phosphatase i n mouse hepatic parenchymal cells. Exptl. Cell Res., 33: 598-601.
Zebrun, W., and H. H. Mollenhauer 1960 Electron microscopic observations on mitochondria
of rat testis fixed in potassium permanganate.
J. Biophys. Biochem. Cytol., 7 : 311-314.
Abbreviations
B.M., basal membrane
D.B., dense body or lysosome
G , Golgi apparatus
Li.,lipids
M., niitochondria
M’., disorganized mitochondria
N., nucleus
N’, granular area interpreted as
remnants of a nucleus
S., Sertoli cell
Sp.A., spermatogonia A
S p . B . , spermatogonia B
Spy., spermatocyte
PLATE 1
EXPLANATION OF FIGURES
20
1
Control mouse. General view of the peripheral part of a germinal
tube. Note at the left two spermatocytes in cytokinesis. X 7,600.
2
Control mouse. Mitochondria of A spermatogonia. X 34,300.
3
Control mouse. Mitochondria of B spermatogonia. X 14,400.
ULTRASTRUCTURE OF IRRADIATED TESTES
J. Hugon and M. Borgers
PLATE 1
PLATE 2
EXPLANATION OF FIGURES
22
x 35,000.
4
Control mouse. Membrane relationship of two Sertoli cells.
5
Control mouse. Three adjoining Sertoli cells.
6
Control mouse. Acid phosphatase reaction in a young spermatid.
Note the lead deposits in the internal cisternae of the Golgi apparatus.
X 32,000.
7
Control mouse. Acid phosphatase reaction in a Sertoli cell. Lead
precipitate in seven dense bodies and two clear vacuoles. x 21,800.
x
37,000.
ULTRASTRUCTURE OF IRRADIATED TESTES
J. Hugon and M. Borgers
PLATE 2
PLATE 3
E X P L A N A T I O N O F FIGURES
24
8
Control mouse. Acid phosphatase reaction. Typical lysosome with
lead salt deposits in a Sertoli cell. x 45,200.
9
Control mouse. Acid phosphatase reaction. Lysosome in a Sertoli
cell with myelin content and lead salt deposits. x 64,350.
10
Control mouse. Acid phosphatase reaction. Lysosome in a Sertoli
cell with vacuolated structures and dense lead precipitate. Note two
lipid areas close to the lysosome. x 45,200.
11
Control mouse. Acid phosphatase reaction. A complex body with
lead precipitate confined to one part of the organelle. X 34,400.
ULTRASTRUCTURE OF IRRADIATED TESTES
J. Hugon and M. Borgers
PLATE 3
PLATE 4
EXPLANATION OF FIGURES
12 Irradiated mouse. Spermatogonia one hour after 400 r. Note the
dilated smooth reticulum and one slightly disorganized mitochondrion
a t M’. The arrows points to a n apparently normal centrosome.
X 17,600.
13 Irradiated mouse. Spermatogonia two hours after 400 r. Disorganized mitochondria, dilatations of the smooth endoplasmic reticulum
and disappearance of spermatogonial membrane between the two
arrows. x 30,100.
14 Irradiated mouse. Spermatogonia four hours after 400 r. Note the
total disappearance of the spermatogonial and Sertoli membranes
beginning at the arrow. X 13,000.
15 Irradiated mouse. Spermatogonia eight hours after 400 r. Two spermatogonia in a n advanced stage of necrosis engulfed in a Sertoli
cell. x 8,250.
ULTRASTRUCTURE OF IRRADIATED TESTES
J. Hugon and M. Borgers
PLATE 4
PLATE 5
EXPLANATION OF FIGURES
16 Irradiated mouse. Acid phosphatase reaction. Sertoli cell with a
great number of dense bodies exhibiting lead s a l t deposits (arrows).
>: 20,300.
17 Irradiated mouse. Acid phosphatase reaction. Part of a Sertoli cell
with a cluster of dense bodies with lead salt precipitate. X 45,000.
28
ULTRASTRUCTURE OF IRRADIATED TESTES
J. Hugon and M. Borgers
PLATE 5
PLATE 6
EXPLANATION OF FIGURES
18 Irradiated mouse. Acid phosphatase reaction. Note the row of lysosomes (dense bodies) with positive reaction along the membrane of
a spermatogonium. X 32,500.
19 Irradiated mouse. Twenty-four hours after 400 r Remnants, of a
spermatogonium engulfed in a Sertoli cell. Note a t the arrow a
dense body. x 30,500.
20
30
Irradiated mouse. Acid phosphatase reaction. Large irregular body
with lead phosphate deposits, interpreted as a residual body. Note
a t the arrow the reaction-positive dense body. X 35,000.
ULTRASTRUCTURE OF IRRADIATED TESTES
J. Hugon and M. Borgers
PLATE 6
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