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Acta Radiologica: Oncology

ISSN: 0349-652X (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ionc19

Long Term Effects of Ionizing Radiation on Mouse


U. Hacker-Klom

To cite this article: U. Hacker-Klom (1985) Long Term Effects of Ionizing Radiation on Mouse
Spermatogenesis, Acta Radiologica: Oncology, 24:4, 363-367, DOI: 10.3109/02841868509136066

To link to this article: http://dx.doi.org/10.3109/02841868509136066

Published online: 08 Jul 2009.

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Acta Radiologica Oncology 24 (1985) F a x . 4





Abstract Material and Methods

The effects of acute or split dose exposure to radiation Male NMRI mice aged 6 to 8 weeks were irradiat-
on murine stem cell spermatogonia were analysed. Flow ed in the testis region with roentgen rays (200 kV,
cytometry was applied to estimate the percentages of
haploid germ cells (round and elongated spermatids) up to 0.5 mm Cu, 0.5 Gy/min) acutely or with a split dose
12 months after irradiation. The recovery in the number of (2 equal doses at 4, 8, 24 or 48 hour intervals). The
haploid germ cells continued gradually during the period anterior part of the body was shielded with lead (0.2
under observation. The intervals between the two equal mm thick). The mice were killed at time intervals
doses in split dose exposures were 0, 4, 8, 24 and 48 after irradiation when the cellular descendants of
hours. Split doses that were 24 h or 48 h apart had more
harmful effects on spermatogenesisthan split doses with 4 the relevant irradiated cells were haploid germ cells.
or 8 hours intervals or acute exposures. The repair capaci- The two testes of 4 mice per dose and time point
ty of the stem cell spermatogonia was remarkably high. were measured separately.
Flow cytometry was performed using a flow cyto-
meter corresponding to that which is commercially
available from Partec AG (Bottmingen, Switzer-
The stem cell spermatogonia are responsible for
land). The cells were isolated and the DNA was
all long term effects of ionizing radiation on fertility.
stained according to the method of ZANTEet coll.
MEISTRICHet coll. (16) applied different assays to
(26, 27) using pepsin and ethidium bromide/mithra-
measure the ability of murine stem cells to regener-
mycin. As the composition of a testis DNA histo-
ate and to repopulate the seminiferous epithelium
gram has been described in detail by ZANTEet coll.
following exposure to radiation, by counting the
(26) and HACKERet coll. (8, 10) it will only be
number of sperm heads, by measuring the level of
explained briefly by the example of Fig. 1b. The first
the germ cell specific isoenzyme LDH-X, and by the
peak (I) in Fig. 1 b represents elongated spermatids
colony assay (1, 14, 25). In the present report, the
and spermatozoa, the second peak (11) round sper-
automated flow cytometric DNA content analysis is
matids, the third peak (111) different germ cells and
proposed as another method for the indirect quanti-
non-germ cells with a 2 c DNA content, and the
tative estimation of the survival of different male
fourth peak (IV) cells with a 4 c DNA content
germ cells, and especially the stem cells, by meas-
(mainly primary spermatocytes and, only to a small
uring the number of cellular descendants (haploid
percentage, (GZ+M)-spermatogonia). Between
germ cells). It has already been proposed that the
peaks Nos I11 and IV, cells synthesizing DNA are
flow cytometric analysis of spermatogenesis could
be used as a sensitive in vivo dosimeter for ionizing
radiation (8, 9, 10). Accepted for publication 23 October 1984.


L b
Fraction ol conlrol


0 7 14 21 28 35
Rcialrve DNA- ~orltcnl Days after rrradiatton

Fig. 1. Photomicrographs of histologic cross sections of testes Fig. 2. Reduction of the number of haploid germ cells during the
and DNA histograms of testicular cell suspensions. Non-irradiat- first 10 weeks after irradiation with 4 different doses. The arith-
ed control mouse (a, b). Mouse 35 days (c, d) and 70 days (e, 0 metic means are plotted. 0.5 Gy (A),2.5 Gy (O), 10 Gy (A), 15
after irradiation with 15 Gy. GY(0).

Since about 80 per cent of the 2 c cells are non-

germ cells (Leydig and Sertoli cells, etc.) (7), which T
I .@+--.-& Control level
are not sensitive to ionizing radiation (3, 6, 12, 13, L

18,20), the 2 c cell percentage is taken as a constant

measure to estimate the radiation-induced reduction 0.5-

of the absolute number of germ cells, instead of the

relative changes in the cellular populations repre-
sented in the histogram. Thus, the percentage of
haploid germ cells was divided by the 2 c cell per-
centage in cases where no absolute cell counts were
performed (10). Calculation of the percentages of c-
0 5 10 15
the different testicular cell types was performed Dose (Gyl
using the cumulative frequency distribution (4, 7, Fig. 3. Dose effect curve of the reduction of haploid germ cells
15). The data were analysed using the Wilcoxon (arthimetic means of the 1 c cell percentages divided by the 2 c
percentages) 70 days after acute irradiation with different doses
rank test. Histologic cross sections were stained (0.1-15 Gy).
according to the method of MEISTRICH et coll. (16),
with PAS/hematoxylin.
ly with 15 Gy 35 days (c, d) and one mouse 70 days
after irradiation (e, f).
The photomicrograph of the histologic cross sec-
Fig. 1 shows 3 DNA histograms (b, d, f) and 3 tion (Fig. 1 a) corresponding to the testis DNA histo-
corresponding photomicrographs of histologic cross gram of a control mouse as described in the section
sections (a, c, e) of the samples from one non- on Material and Methods shows tubuli contorti in
irradiated control (a, b), one mouse irradiated acute- different stages of spermatogenesis. In Fig. l c , a

Fraction of conlrol
and undifferentiated spermatogonia 35 days after
irradiation, and most marked after irradiation of
differentiating spermatogonia 28 days after irradia-
tion. Following exposure of stem cell spermatogonia
(70 days after irradiation), the number of haploid
germ cells was not much reduced.
Fig. 3 shows the dose-effect relationship for the
reduction of the number of haploid germ cells 70
days after irradiation. This dose-effect curve had a
0.1- shoulder. Only exposure to more than 2.5 Gy led to
0 3 6 12 a remarkable decrease in the haploid germ cells.
Months after irradiation In Fig. 4, the arithmetic means of the 1 c cell
Fig. 4. The arithmetic means of the 1 c cell percentages divided numbers (% 1 c/% 2 c) at different intervals after
by the 2 c cell percentages were plotted in time-dependence 3, 6 acute exposures with 5 different doses are plotted.
and 12 months after acute exposures with 5 different doses. 3 Gy
(A), 4.5 GY( W , 6 GY(O),9 GY (V), 12 Gy (0. This figure shows an extension of the time scale in
Fig. 2. Three months after irradiation, there was a
Fraction of conlrol
clear dose-dependence in the germ cell reduction.
Six and 12 months after irradiation, however, a
I Control /eve/
strong dose-dependence was no longer seen. The
repopulation continued gradually during the 12
months of the observation period. The regeneration
and repopulation capacity of the stem cells was
surprisingly high, although following exposure to
more than 4.5 Gy the control level was not com-
pletely reached.
In Fig. 5 , the arithmetic means of the 1 c cell
numbers (% 1 c/% 2 c) were plotted after two split
0 4 8 24 48

Interval fhj
dose exposures of a total of 12 Gy at 0 , 4 , 8 , 2 4 or 48
Fig. 5. The arithmetic means of the 1 c cell percentages divided hour intervals between the two equal exposures of 6
by the 2 c cell percentages were plotted in a time-dependent Gy. Following a split dose exposure at 24 or 48 hour
manner following split-dose exposures of 12 Gy at 0 , 4 , 8 , 2 4 or 48
hour intervals. 3 (A), 6 (O), 12 (0)months after irradiation. intervals, the damage from the 12 Gy absorbed radi-
ation dose to the stem cell spermatogonia obviously
was heavier than after split dose exposures at 4 or 8
depletion of the seminiferous epithelium from germ hour intervals or an acute exposure of 12 Gy. This
cells 35 days after irradiation is seen; in the corre- was significant for all values 3 months after irradia-
sponding DNA histogram (Fig. 1 d) peaks Nos I and tion and for most values 6 and 12 months after-
I1 representing haploid germ cells are missing. Fig. wards.
1 e demonstrates that 70 days after irradiation with No great difference was noted in the long term
15 Gy the interstitial and Sertoli cells were relatively damage from the 12 Gy split dose exposures at 0,4,
increased because of a reduction of germ cells. In or 8 hour intervals. However, following 12 Gy split
the DNA histogram of Fig. 1 f the haploid germ cells dose exposures at 24 or 48 hour intervals, the long
were reduced 70 days after the irradiation. The 2 c term damage 12 months after irradiation was heav-
cells were relatively increased when compared with ier. Following all 12 Gy exposures, the regeneration
Fig. l b . and repopulation during 12 months remained incom-
Fig. 2 shows the pattern of reduction of the num- plete in relation to the control level.
ber of haploid germ cells (round and elongated sper-
matids and spermatozoa, respectively) during the
first 10 weeks after acute irradiation with 4 different
doses: 0.5, 2.5, 10 and 15 Gy. The reduction was The regeneration capacity of the murine stem
moderate after irradiation of spermatids, spermato- cells and their ability to repopulate the seminiferous
zoa and spermatocytes (2-21 days after irradiation) epithelium after exposure to radiation is surprising.

Even after exposure to more than 10 Gy, that is ysis of the effect of chronic whole-body irradiation
about twice as much as the dose inducing permanent with gamma-rays on the spermatogenic elements and
sterility in man (20), mouse spermatogenesis recov- the interstitial tissue of the testes of mice. J. nat.
Cancer Inst. 9 (1948), 133.
ers and enables fertility (16). Fertility is regained if 4. GOHDEW., SCHUMANN J., BUCHNERTH., OTTOF.
the testicular sperm concentration is 15 per cent of and BARLOGIE B.: Cytophotometry application in tu-
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begin to differentiate before increasing, thus reduc- Hodentumors. Strahlentherapie 153 (1977), 257.
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acute exposure to 4 Gy in man, it takes 5 years or Strahlung am Model1 der Spermatogenese der Maus.
even more before the original sperm concentration Inaugural-Dissertation im Fachbereich Biologie. Uni-
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8. - SCHUMANN J. and GOHDEW.: Effects of acute
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a complete regeneration of the seminiferous epithe- flow cytometry. Acta radiol. Oncology 19 (1980), 361.
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