Direct cell kill and inhibition of mitosis have been suggested as mechanisms to explain the occurrence of absolute sterility following the irradiation of the testes. In order to obtain information on the existence and dose dependency of the mechanisms for man, a controlled study was initiated. Sixty-four  men received a single midorgan dose to both of their testes ranging from 7.5 to 400r (f = .95). It was deduced from resulting pre-sterile period and sterile period data that both cell kill and mitosis halting mechanisms were operating. The maximum observed sterile period was 501 days with eventual recovery observed in each individual where the follow-up was complete. Thus man appears to be highly radiosensitive in regard to temporary sterility but quite radioresistant in regard to permanent sterility

Paulsen, C. A.

Thorslund, T. W.

English

NASA Technical Reports Server

EFFECTS OF X-RAY IRRADIATION ON HUMAN SPERMATOGENESIS
T.W. Thorslund and C.A. Paulsen, (University of Washington)
Direct cell kill and inhibition of mitosis have been suggested as mechanisms
to explain the occurrence of absolute sterility following the irradiation of the
testes. In order to obtain information on the existence and dose dependency of the
mechanisms for man, a controlled study was initiated in 1963. A total of 209 normal
adult non-Catholic inmate volunteers at the Washington State Penitentiary in Walla
Walla participated in the experiment. Sixty-four of the men received a single mid-
organ dose to both of their testes ranging from 7.5 to 400r (f - .95). The remaining
men served as various control groups. Testicular function was evaluated primarily on
the basis of weekly seminal fluid examinations where the response or end-point was
taken to be azoospermia or complete sterility. In order to obtain direct information
on testicular morphological changes, periodic unilateral testicular biopsies were also
performed on a number of individuals in both theexposed and non-exposed groups.
It was deduced from the resulting length of the pre-sterile period and sterile
period data that both cell kill and mitosis halting mechanisms were operating. Estimates
of ED50 of 75r and 27r for cell kill and mitosis halting, respectively, were obtained.
The maximum observed sterile period was 501 days with eventual recovery observed in
each individual where the follow-up was complete. Thus man appears to be highly radio-
sensitive in regard to temporary sterility but quite radioresistant in regard to
permanent sterility.
Various mechanisms suggested by animal
experiments have been postulated to ex-
plain the phenomenon of absolute steril-
ity or azoospermia resulting from irra-
diation of the testes.
Oakberg (ref. i) attributes the
observed depletion or total absence of
germ cells in the epithelium of the
seminiferous tubules to be due to direct
cell kill. Under thishypothesis the
length of the pre-sterile period or time
to first azoospermia would be determined
by the time required for the resistant
cells to complete development and reach
the ejaculate. The duration of azoosper-
mia or length of the sterile period is
considered to be made up of two compo-
nents. The duration of the first compo-
nent is equivalent in length to the age
of the youngest cohort of germ cells
that was not totally decimated. The
second component of the sterile period
is the time required for the surviving
stem cells to multiply sufficiently to
reach a critical density or cluster size
necessary for differentiation to take
place.
Jones (ref. 2) postulates that an
inhibition of mitosis may be the mech-
anism responsible for the Sterile period.
Under this hypothesis the time to first
azoospermia would be equivalent to the
total maturation time plus the time
post-irradiation required for mitosis
to be halted. The duration of the
sterile period is identical to the time
required for the mitotic mechanism to
reDair itself.
The existence and dose dependency of
the postulated mechanisms with respect
to human beings is of obvious scientific
interest and growing medicolegal impor-
tance as human exposure to radiation
increases. The variability in sperma-
togenic kinetics observed among various
species in regard to their gonadal re-
Sponse to dfiferent levels of radiation
(refs. 3 to 5) renders any human
sensitivity estimates based on animal
data highly questionable.
NORMAL HUMAN SPERMATOGENIC KINETICS
In recent years a series of experiments
were performed that allow one to estimate
the time required for a human germ stem
cell to mature and be passed into the
ejaculate.
Clermont (ref. 6), Heller and Clermont
(ref. 7) have devised a highly explicit
model for the mode of development and
timing of human spermatogenesis. The
mode of development portion of the model
is based on the observed ratios of germ
cell types in various stages of matura-
tion counted in biopsy specimens. The
timing relationships were determined by
using radioautographs of testicular sec~
tions following intratesticular injections
of H3-thymidine. The maximum transport
time of the mature spermatozoa from their
release from the Sertoli cell cytoplasm
into the ejaculate has been determined
by Rowley et. al. (ref. 8) to be no more
than twenty-one days. Freund and Davis
(ref. 9) observed a thousand fold de-
pletion of sperm count at the fifth
ejaculation post vasectomy. Thus the
time required to evacuate ductal storage
of spermatozoa is virtually equivalent
to the time to the fifth ejaculation.
229
https://ntrs.nasa.gov/search.jsp?R=19720009980 2020-03-17T03:50:20+00:00Z
Theminimumtime required For the mature
spermatozoato reach the ejaculate under
certain conditions is considered to be
less than oneday.
A model for the maximumtime required
for a maturating humangermstem cell to
reach the ejaculate basedon the preced-
ing considerations is presented in a
schematicmannerin figure i. This
modelwill be utilized to test the
plausibility of the various suggested
disrupting mechanismsin regard to human
spermatogenesis.
nGUEl MODEL FOR MODE OF DEVELOPMENT AND TIMING
OF HUMAN SPERMATOGENESIS
Time From New Ad (doys)
_12.7_31.2_39.9_63,9_ 863 _107.3 _142_3
I I I i i I
/ / Component Time Intervols (doys) /
-12 7-,-P,-- 18.5 -_,.-_8.7 -,._-q_- 2410 _ 22.4 _ 2L0-_--,_55.0/ / / / I Mox_r.m I Mox...m
T . port Ei_otio.
Stem Ap or 8 PI So Spermotozoon Ouctol Into
Cell Ad Storoge EjoculoteAd
Abhrevl_,lnn_ A._, A dark s;'ermato_osl3: A9, A pa[_ S_ll;,_h,_,_,,_o; B. B
HUMAN EXPERIMENT OF THE EFFECT
OF X-RAYS
In order to obtain a reliable estimate
of the existence and dose dependency of
the postulated spermatogenesis disrupting
mechanisms for man, a controlled experi-
ment was undertaken in 1963. This study
utilized a group of 209 healthy non-
Chatholic inmate volunteers at the
Washington State Penitentiary in Walla
Walla. Each inmate selected for the
study had to express a desire for a
vasectomy. This was to be performed
at the completion of his involvement
in the study. The inmate and, if married,
his wife both signed a written consent
form authorizing all experimental pro-
cedures.
Extensive pre-irradiation sperm samples
were obtained by masturbation for each
inmate and sperm density estimates of
ejaculate made to demonstrate normal
spermatogenesis. The testes of sixty-four
of the men received a mid-organ dose of
X-rays ranging from 7.5 to 400.r. (f
factor = .95) administered acutely by a
Maximar 250 Kv unit. The remaining 145
men served as various types of controls.
In addition, in order to obtain direct
information on testicular morphological
changes, periodic unilateral testicular
biopsies were performed on a number
of individuals in both the irradiated
group and non-irradiated group. Weekly
sperm samples were collected post-
irradiation and sperm density estimates
made even though the end point of central
interest was azoospermia.
The number of inmates in each biopsy-
radiation group and the number of these
that were observed to have achieved
azoospermia by 142 and by 210 days is
shown in table i. The duration in days
of the pre-sterile period and the sterile
period for each inmate in his biopsy-ra-
diation group that became azoospermic
is given in table 2. The question marks
(?) indicate that the individual was
lost for observational purposes while
still azoospermic.
Toble I
DATA USED TO ESTIMATE PARAMETERS IN POSTULATED M(MM_LS
X-Roy Number of Froction Sterile Fmctioe Sterile
(r) Biopsies by 142 Ooys by 210 Ooys
400 0 2/2 2/2
I I/I I/I
2 2/2 2/2
,00 0 _/7 7/7/9 9/9
3 2/2 2/2
50 0 I/8 5/R
Lo U 0/5 0/5
0 0 0/96 0/96
I 0/29 0/29
2 1/18 1/18
3 0/I 0/I
Toble 2
TIME SEQUENCE OF AZOOSPERMIA AS A FUNCTION
OF RADIATION AND BIOPSIES
X-Roy Number of Pre-Sterile Sterile
(r) Biopsies Period in Doys Period in Doys
230
o
3o
5o
IOO
IOO
IOO
IOO
4oo
4oo
4oo
38 ?>509
95 182
194 7
115 70
160 91
171 84
195 21
202 49
88 209
107 91
129 112
141 112
141 112
t52 61
189 112
65 108
I00 421
I09 ?>42
III ?>7
113 112
I 14 252
124 ?>152
201 ?> 21
209 62
145 327
127 291
127 501
76 147
98 196
I 15 329
113 338
t 16 342
The 142 day time period was selected
since it represents the maximum pre-
sterile period possible if the mechanism
causing sterility is complete cohort
decimation. This estimate is based on
the observation that if an inmate mastur-
bated only at his prescribed collection
time evacuation would be accomplished in
7 x 5 = 35 days. Thus the maximum pre-
sterile period is 35 + 21 + 86 = 142 days.
The 210 day time period was selected since
no new occurrences of azoospermia were
observed after that time.
MODELS FOR THE EFFECTS OF X-RAYS
From figure 1 we note that a pre-sterile
period of less than 86 days can only be
explained in terms of the postulated
mechanisms by assuming death of a cohort
of differentiating germ cells. For two
inmates azoospermia occurred at 65 and 76
days confirming the existence of that
mechanism. If the pre-sterile period was
greater than 142 days then complete co-
hort decimation did not occur. A lower
limit on the dose dependency of the
cohort decimation effect can be determined
by assuming that if azoospermia occurs by
142 days the effect occurred. Thus the
observed fraction of inmates becoming
azoospermic by 142 days shown in table 1
can be utilized to estimate the EDs0 of
the effect.
A model relating the probability of
total cohort decfmation to the dose of
X-rays can be derived under the following
assumptions:
i) The probability of the most
sensitive cell type being killed
given a dose of X-rays of size x
follows a one-hit-curve of the
form
P = (i - e-eX).
ii) The cohort size of the most
sensitive type of germ cell of
an individual is a random variable
N which has a poisson distribution
with parameter _.
iii) A person is azoospermic if all N
of his most sensitive type of cell
are killed.
Under these assumptions the probability
of total cohort decimation is
= -_e -@x
P(x) = [ (i - e-SX) N e-_ N_ e
N=0 N!
and the dose of radiation that results in
50% of the population becoming azoospermic
due to cohort decimation is
__e-OXs0
1/2 = e
or
xs0 = EDs0 = [-in(In 2) + In _]/0
The dose required (LDs0) to kill 50% of
the most sensitive type of germ cell can
also be estimated from this model. It
follows directly from the assumption of
a one hit curve for cell kill that
-0Xs0
1/2 = 1 - e
or
Xs0 = LDs0 = in 2/8
The parameters of the model (I,8) were
estimated from the observed data by the
maximum likelihood method (ref. i0), ad-
justing for the probability that a
testicular biopsy could also independently
couse azoospermia by 142 days. The
estimate of EDs0 was found to be 74.6r
with a 95% confidence interval obtained
by using Fieller's Theorem (ref. ii) of
57.2 to i03.6r. In addition, it is of
interest to note that this rather indi-
rect method of estimating the LDs0 of
cell kill gives an estimate of 23.2r
which is in remarkable agreement with
Oakberg's estimate (ref. 12) of an LDs0
of between 20 and 24 rad of X-ray for
spermatogonia.
The longest observed pre-sterile period
was 209 days with i0 out of the 32 in-
mates who became azoospermic having a
pre-sterile period greater than 142 days.
The maximum observed sterile period was
501 days where a maximum of 142 - 127
= 15 days could be explained by cohort
decimation. To explain these results by
the cell kill hypothesis several addi-
tional assumptions are needed. We need
to assume that some stem cells are
capable of functioning normally for at
least 209 - 142 = 67 days post-irradia-
tion and then die. For one individual
we must assume that the total time where
stem colony growth took place if 501 - 15
= 486 days and thus the number of doubl-
ings was 486/12.8 = 38 and the total
restored stem cell population for an
individual tubule is at least 238 before
differentiation took place. Neither of
these assumptions appear to be very
tenable.
Oakberg (ref. 13) suggests upon viewing
similar results of length of the sterile
period for humans based on a preliminary
report by Heller (ref. 14) that differ-
entiation of the stem cells produces
non-viable daughter cells causing the
stem cell population to build up slowly
thus prolonging the sterile period.
However, on inspection of our testicular
biopsies (ref. 15) we observe a period
of very low stem cell density followed
by a rapid doubling of stem cells in line
with the 12.8 day doubling time, which in
turn is followed by the differentiation
of the stem cells and eventual recovery.
In the light of these results it appears
reasonable to assume that mitotic divi-
sion is also halted in some manner by
the radiation.
231
If we assumethat individuals have a
normal distribution of log dose toler-
anceswith regard to somemitotic divi-
sion mechanism,then the probability of
azoospermiagiven a log dose of radia-tion of size x is to a very close
approximation 1
P(x) = -(e+_x)1 + e
and -_/8Xs0= EDs0= e
If we assume that a critical volume
in the testes exists that has an over-
riding control over the mitotic division
mechanism and is damaged with one hit,
or if we assume that tolerances to dose
have the exponential distribution, then
the
-0x
P(x) = 1 - e
and
xs0 = EDs0 = in 2/@.
Estimates of the parameters (_, B) or
(8), for these models was obtained by
maximum likelihood estimation adjusting
for the probability of a biopsy causing
sterility, using as data the fractions
of individuals who became azoospermic by
210 days. Estimates of EDs0 were 36.3r
with a 95% confidence interval of 19.8
to 50.3r for the normal distribution of
log dose tolerance model and 27.2r with
a 95% confidence interval of 18.8 to
48.8r for the one hit model. The varia-
tion in the confidence intervals for
these two models are minimal while the
differences in the EDs0 estimates are
primarily due to different dose scales.
A summary of the ED50 results is shown
in table 3.
Simulation experiments were run on
these models to verify that the asymp-
totic approximations used to obtain the
confidence intervals were valid for the
sample sizes used in the experiment. In
all cases a close agreement was obtained
between the observed and expected results
which virtually assures the statistical
validity of the stated intervals.
CONCLUSIONS
In men the occurrence and length of
azoospermia or complete sterility
following X-ray irradiation is made up
of a number of components. It appears
reasonable to assume that complete cohort
decimation of some germ cell type does
take place following exposure to suffi-
ciently large doses. However, doses that
are not large enough to cause total
cohort decimation are still able to stop
spermatogenesis by halting mitosis of
the surviving stem cells for extended
periods of time needed for repair. The
repaired stem cells multiply until a
critical cluster size is reached after
which differentiation occurs followed by
the reappearance of sperm in the ejaculate.
Man also appears far more sensitive to
radiation than the rodent who needs al-
most ten times the dose to become
azoospermic. The time needed for man to
recover is also much longer than would
be expected from animal experiments,
ranging up to a year and a half after
exposure. However, the evidence does
point to eventual recovery even for doses
of 400_.
In short, man is very radiosensitive in
regard to temporary sterility periods as
long as one and a half years but very
radioresistant to complete sterility.
ACKNOWLEDGEMENTS
Supported in part by AEC Contracts AT(45-I)-1781
and AT(45-I)-2225 and NIH grants AM 05161 and
AM 05436.
The authors wish to thank Donald J. Moore for
his expert technical assistance.
Toble 3
EDso ESTIMATES FOR POSTULATED MODELS
AND ITS 95% CONFIDENCE LEVEL
Model Doys Post Lower Upper
P (x) Irrodiotion Limit ED5o r. Limit
e -ke-ox 142 5Z2 74.6 103.6
I
i +e-(Cc+ _ln x) 210 19.8 36.3 50_3
I - e -ex 210 18.8 27.2 48.8
232


Effects of X-ray irradiation on human spermatogenesis

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