Acta Radiologica: Oncology

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Long Term Effects of Ionizing Radiation on Mouse

Spermatogenesis

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

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

FROM THE CLINIC O F RADIATION BIOLOGY, UNIVERSITY OF MUNSTER, D-4400 MUNSTER, WEST GERMANY.

LONG TERM EFFECTS OF IONIZING RADIATION ON MOUSE

SPERMATOGENESIS

U. HACKER-KLOM

Abstract Material and Methods

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

363
364 U. HACKER-KLOM

L b
Fraction ol conlrol

l------
d

I
0 7 14 21 28 35
.
n
70
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
the different testicular cell types was performed
using the cumulative frequency distribution (4, 7,
15). The data were analysed using the Wilcoxon
rank test. Histologic cross sections were stained
according to the method of MEISTRICH et coll. (16),
with PAS/hematoxylin.
Results
Fig. 1 shows 3 DNA histograms (b, d, f) and 3
corresponding photomicrographs of histologic cross
sections (a, c, e) of the samples from one non-
irradiated control (a, b), one mouse irradiated acute-
0.1-
c-
0 5 10 15
Dose (Gyl
Fig. 3. Dose effect curve of the reduction of haploid germ cells
(arthimetic means of the 1 c cell percentages divided by the 2 c
percentages) 70 days after acute irradiation with different doses
(0.1-15 Gy).
ly with 15 Gy 35 days (c, d) and one mouse 70 days
after irradiation (e, f).
The photomicrograph of the histologic cross sec-
tion (Fig. 1 a) corresponding to the testis DNA histo-
gram of a control mouse as described in the section
on Material and Methods shows tubuli contorti in
different stages of spermatogenesis. In Fig. l c , a
 IONIZING RADIATION ON MOUSE SPERMATOGENESIS
Fraction of conlrol
0.1-
I I
0 3 6 12
Months after irradiation
Fig. 4. The arithmetic means of the 1 c cell percentages divided
by the 2 c cell percentages were plotted in time-dependence 3, 6
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.
Fraction of conlrol
I Control /eve/
0.7{
0 4 8 24
~
Interval fhj
Fig. 5. The arithmetic means of the 1 c cell percentages divided
by the 2 c cell percentages were plotted in a time-dependent
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.
depletion of the seminiferous epithelium from germ
cells 35 days after irradiation is seen; in the corre-
sponding DNA histogram (Fig. 1 d) peaks Nos I and
I1 representing haploid germ cells are missing. Fig.
1 e demonstrates that 70 days after irradiation with
15 Gy the interstitial and Sertoli cells were relatively
increased because of a reduction of germ cells. In
the DNA histogram of Fig. 1 f the haploid germ cells
were reduced 70 days after the irradiation. The 2 c
cells were relatively increased when compared with
Fig. l b .
Fig. 2 shows the pattern of reduction of the num-
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
moderate after irradiation of spermatids, spermato-
zoa and spermatocytes (2-21 days after irradiation)
365
I
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
shoulder. Only exposure to more than 2.5 Gy led to
a remarkable decrease in the haploid germ cells.
In Fig. 4, the arithmetic means of the 1 c cell
numbers (% 1 c/% 2 c) at different intervals after
acute exposures with 5 different doses are plotted.
This figure shows an extension of the time scale in
Fig. 2. Three months after irradiation, there was a
clear dose-dependence in the germ cell reduction.
Six and 12 months after irradiation, however, a
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
48
dose exposures of a total of 12 Gy at 0 , 4 , 8 , 2 4 or 48
hour intervals between the two equal exposures of 6
Gy. Following a split dose exposure at 24 or 48 hour
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
hour intervals or an acute exposure of 12 Gy. This
was significant for all values 3 months after irradia-
tion and for most values 6 and 12 months after-
wards.
No great difference was noted in the long term
damage from the 12 Gy split dose exposures at 0,4,
or 8 hour intervals. However, following 12 Gy split
dose exposures at 24 or 48 hour intervals, the long
term damage 12 months after irradiation was heav-
ier. Following all 12 Gy exposures, the regeneration
and repopulation during 12 months remained incom-
plete in relation to the control level.
Discussion
The regeneration capacity of the murine stem
cells and their ability to repopulate the seminiferous
epithelium after exposure to radiation is surprising.
366 U . HACKER-KLOM
Even after exposure to more than 10 Gy, that is
about twice as much as the dose inducing permanent
sterility in man (20), mouse spermatogenesis recov-
ers and enables fertility (16). Fertility is regained if
the testicular sperm concentration is 15 per cent of
the control level (16). In man, the regeneration ca-
pacity of the stem cell spermatogonia is much lower
(5, 11, 21, 22, 24). In mice, the stem cell spermato-
gonia undergo mitosis until they have reached about
their original number (18); in man, the stem cells
begin to differentiate before increasing, thus reduc-
ing their number a second time. This phenomenon
induces a second depletion of the seminiferous epi-
thelium, thus increasing the radiation sensitivity of
the human exocrine testicular function. After one
acute exposure to 4 Gy in man, it takes 5 years or
even more before the original sperm concentration
in the ejaculate is reached (22). This corresponds to
about 30 spermatogenetic cycles (19). In the mouse,
a complete regeneration of the seminiferous epithe-
lium is seen already 3 months after the same radi-
ation dose (2). This corresponds to about three cy-
cles of spermatogenesis (17). However, following all
acute exposures to radiation of more than 4.5 Gy,
the regeneration remains incomplete during the 12
months following the irradiation. This result agrees
with the data of MEISTRICHet coll. (16). According
to RUGH (23), an exposure to 16 Gy destroys the
stem cell spermatogonia completely, thus inducing
permanent sterility. The method proposed in this
report, using flow cytometry, is a fast way to quanti-
fy the spermatogenetic function.
. ACKNOWLEDGEMENTS
The investigation was supported by the Commission of
the European Communities for Biology, Radiation Protec-
tion and Medical Research, contract number BIO-E-538-
D (B). Thanks are also due to Mrs H. Zold and Mrs P.
Berkes for skilful technical assistance.
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