Mutation Research, 172 (1986) 255-263

255

Elsevier
MTR 01118

Micronucleated spermatids in the seminal fluid of smokers and nonsmokers

Jaana L~ihdetie
Mutagen Laboratory, Department of Industrial Hygiene and Toxicology, Institute of Occupational Health, Topeliuksenkatu 41 a A,
SF-00250 Helsinki (Finland)

(Received15 January1986)
(Revisionreceived27 June 1986)
(Accepted4 July 1986)

Summary
This study presents a novel approach to the analysis of chromosome damage in human male germ cells.
Round spermatids are present at a low frequency in seminal fluid, and meiotic chromosome breakage may
be observed in these cells by analyzing micronuclei (MN). Semen samples from 68 men, including 62
subfertile men and 6 fertile donors, were analyzed. Preparations were made of the round cell types in the
semen and after PAS-hematoxylin staining, the number of MN in 100 Golgi-phase or cap-phase
spermatids was scored per man. The frequencies of MN were 1.15 1.42% in the smoking subfertile men
and 0.82 1.30% in the nonsmoking subfertile men. The difference was not statistically significant. When
the smokers were divided into groups according to the number of cigarettes smoked per day, no significant
differences were observed compared to the nonsmokers. Neither was an effect of smoking observed when
the time smoked in years was taken into account. The frequency of MN in ejaculated spermatids in human
males was observed to be considerably higher than that reported for testicular spermatids of unexposed
rodents.

Analysis of micronuclei (MN) is possible in

several proliferating tissues, including the bone
marrow and testis, and is a rapid and simple test
for chromosome damage compared to metaphase
chromosome analysis. In rodents, clastogenic
agents such as X-rays (L~ibdetie and Parvinen,
1981; Tates, 1984), fission neutrons (Tates et al.,
1983; Tares, 1984), adriamycin (LS.hdetie, 1983a),
cyclophosphamide and dimethylbenzanthracene
(LS.hdetie, 1983b), mitomycin C (Tates et al., 1984)
and ethylnitrosourea (Tates et al., 1985) have been
shown to increase the frequency of MN in early
spermatids. Agents inducing spindle dysfunction
do not seem to give rise to MN in the testis
(L~ihdetie and Parvinen, 1982; Tates et al., 1983).
Ultrastructural studies have shown that MN arise
during meiotic divisions from lagging chro-

mosomal material and are distinct from signs of

cellular death, e.g. autophagocytic vacuoles
(L~ihdetie et al., 1985). In humans, MN have previously been analyzed in bone-marrow cells, cultured lymphocytes, fibroblasts and buccal mucosal
cells. The use of MN as an indicator of genotoxic
exposure in man has been studied by H/Sgstedt
(1983).
The present study was undertaken to analyze
MN in round spermatids known to exist in various quantities in human ejaculate (Couture et al.,
1976; Sperling and Kaden, 1971; Sun and White,
1979). The meiotic micronucleus method developed by Tates et al. (1983) for testicular material
of mammals, including man, was applied to the
analysis of ejaculated cells. The identification of
various round cell types in the seminal fluid is

0165-1218/86/$03.50 1986 ElsevierSciencePublishersB.V.(BiomedicalDivision)

256
described. The effect of smoking habits on the
frequency of MN in spermatids was analyzed statistically. The results were also compared to earlier
data on rodent spermatids.
Materials and methods
Semen samples were obtained from 68 men; 62
of them attended the Andrological Laboratory of
the Institute of Biomedicine, University of Turku,
because of difficulties the couple were experiencing in achieving a pregnancy, a n d t h e term "subfertile" refers to them in this article. Of the 6
voluntary semen donors, 5 were fathers of small
children and 1 had unproven fertility. They are
referred to as "fertile" in this article. A questionnaire containing, among others, questions about
the duration of abstinence before the sample was
taken, smoking habits, alcohol consumption, occupational exposures, diseases and therapeutic
drugs was filled in by the subjects at home and
brought to the laboratory together with semen
samples taken within 2 h after ejaculation.
A complete semen analysis was performed.
Sperm count and percentage of motile sperm were
determined using a hemocytometer. Smears were
made of fresh semen, air-dried and fixed in
ethanol-ether 1:1 (v/v) for 30 min. They were
stained with the Papanicolaou technique (Belsey
et al., 1980), scored at 400-fold magnification and
100 spermatozoa were morphologically classified
to determine the percentage of abnormal forms.
The same slides were also used for MN analysis,
but with poor results.
To improve the yield of "round cells" for MN
analysis, a differential centrifugation technique
using at least 1.0 ml of semen and 5.0 ml of testis
isolation medium (TIM) (Tates et al., 1983) was
applied. The sample was mixed thoroughly,
centrifuged at 400 rpm for 5 min and the pellet
resuspended in 5.0 ml of TIM. This step was
repeated twice. Before removing the supernatant,
the sample was left to stand for 30 min allowing
motile spermatozoa to swim away from the pellet.
The numbers of round cells and sperm during
different steps of the separation process were determined by hemocytometer counting in 6 randomly selected samples. The resuspended pellet
was placed in 3 drops on a clean slide, and the

cells allowed to form a sediment for 15 min. The

slides were then carefully covered with Zenker-formol fixative; the fixation time was 60 min. The
slides were rinsed with 70% ethanol and stored in
it until staining with PAS-hematoxylin. After a
change into distilled water, the slides were immersed in 1% periodic acid for 10 min and rinsed
with distilled water. They were treated with Schiff
reagent for 30 min and rinsed for 15 min in running tap water. Counter-staining with Mayer's
hemalum was done for 10 min followed by differentiation in HCl-ethanol. After rinsing in running
tap water for 10 min, the slides were dehydrated
in ethanol, cleared in xylene and mounted with
Depex.
100 round spermatids of the Golgi phase or cap
phase were scored at 1000-fold magnification and
the number of MN in them counted. Fig. 1 shows
2 typical round spermatids of slightly different
size and 2 similar cells with MN. Identification of
the spermatid stages was essentially based on the
descriptions for testicular cells given by Clermont
and Leblond (1955). The criteria for identification
of other cell types in the ejaculates are described
later. If uncertainty existed, the cell was not scored
for MN. Multinucleate spermatids were quite often
seen but cells with more than 2 nuclei were not
scored. Neither were round spermatids showing
heavy signs of degeneration (nuclear condensation
and hyperchromatic staining of the nucleus)
scored. The MN had to be round or oval, separate
from the main nucleus and have the same staining
characteristics as the main nucleus. The proportions of the total number of leukocytes and the
total number of spermatids were counted in each
slide.
Statistical analyses included comparisons of the
mean values of the 3 groups: fertile men, nonsmoking subfertile men and smoking subfertile
men by one-way analysis of variance followed by
a pairwise comparison using Student's t test for
age, abstinence time, semen volume, percentage of
abnormal sperm and percentage of motile sperm,
and the Mann-Whitney U test for sperm density,
total sperm count and frequency of MN. When
the men were grouped into 4 groups according to
the number of cigarettes smoked per day, comparison of the means of MN frequencies was done
by the Mann-Whitney U test. Finally, regression

257

~i i, ~ i~,~~ i~ ~ ~~
~i ~ ~ii !ii i iiili,/iii~ i iii ii

Fig. 1. Two typical early spermatids of slightly different size (A and C) and two corresponding spermatids with micronuclei (B and
D). The bar indicates 5/~m.

analysis was performed using the pack-year concept (packs of cigarettes smoked per day years
smoked) and a square-root transformation of M N
values. A S U R V O 76 c o m p u t e r p r o g r a m package
(Mustonen and Mellin, 1980) was used for the
calculations.

Results
The quantity of " r o u n d cells" in the resusp e n d e d pellet was variable, representing a recovery of 28.4 _ 15.6% (mean _ S.D., N = 6) of
the original n u m b e r of these cells in the semen
sample. 95-100% of the spermatozoa were rem o v e d by the differential centrifugation.
The m o r p h o l o g y of typical cell types in the

258
ble to classify due to degenerative changes. Since
spermatids are not in their physiological environm e n t when present in the ejaculate, various degrees of degeneration of the cells can be observed.
This causes a slight variation in the diameters of

preparations is shown in Fig. 2. T h e round

spermatids represented a minority of these cells in
the ejaculate, the majority usually consisting of
later spermatid stages (acrosome-phase and m a t u ration-phase spermatids) and spermatids impossi-

:i)(i//ii IIIIiiii i!ii!i

~i

!ii!~iiii!ii
~i~)i~i!i~i~ii
~ ~ii~

ili

i~ii~....

iii :i

iiiii

Fig. 2. Examples of cell types observed in the PAS-hematoxylin-stained preparations. See text for further details. (A) Golgi-phase
spermatid; (B) binucleate Golgi-phase spermatid; (C) cap-phase spermatid; (D) acrosome-phase spermatid; (E) spermatocyte in
meiotic prophase; (F) spermatocyte arrested in meiotic metaphase with condensed chromosomes; (G) polymorphonuclear leukocyte;
(H) 2 lymphocytes; (I) squamous epithelial cell. The bar indicates 5/tm.

259

the nuclei and the sizes of the round spermatids.

The PAS staining of the acrosome system of the
spermatids was variable and rather weak compared to that described for testicular cells (Clermont and Leblond, 1955), presumably also because of differences in the degree of degeneration.
Golgi-phase spermatids were identified by a
bluish nucleus with a few granules of heterochromatin, while the cytoplasm was pale (Fig. 2A).
Many of them contained a weakly PAS-positive
area (the idiosome) or 1 or 2 PAS-positive granules (proacrosomic granules or acrosomic granules) in the cytoplasm. In cap-phase spermatids
the acrosome was usually visible as a PAS-positive
border or as a pale, flattened vacuole on the
surface of the nucleus (Fig. 2C). In acrosome-phase
spermatids the nucleus protruded towards the periphery of the cell (Fig. 2D). Maturation-phase
spermatids and mature spermatozoa were identified by their small, typically formed head and the
presence of a tail.
Spermatocytes were recognized by their larger
size and a coarse meshy chromatin (Fig. 2E) which
was maximally condensed during metaphase (Fig.
2F). Spermatogonia were not observed in the
specimens.
The vast majority of leukocytes observed were
polymorphonuclear cells. They were easily identified by the bright purple color of the cytoplasm
and lobulation of the nucleus (Fig. 2G). In some
cases the proportion of granulocytes of all round
cells in the semen was very high, indicating an
infection in the reproductive tract; 29% of the
subfertile men had more leukocytes than spermatids in their semen samples. Lymphocytes were
recognized by their small diameter, scanty cytoplasm and either a kidney-shaped nucleus or an
indentation in the nucleus (Fig. 2H). The dark and
lumpy consistence of lymphocyte nuclei was clearly
different from that of round spermatids. Macrophages were large cells with a bean?shaped
nucleus and PAS-positive granules in the cytoplasm; they often contained phagocytized sperm
as described by Phadke and Phadke (1961).
Squamous epithelial cells had a very large diameter and a polygonial shape (Fig. 2I), while
cells from the transitional epithelium of the urinary
tract had large, oval nuclei, PAS-positive cytoplasm and were often observed in clumps. Ob-

servations of these epithelial cells were rather uncommon.

An attempt was made to count MN in ordinary
seminal smears stained by the Papanicolaou
technique. However, this was practically impossible since only smears from semen samples with
high numbers of immature testicular cells (initial
number of "round ceils" at least 1 million/ml
semen) yielded 100 well-identified round spermatids and their scoring was very slow.
9 men were excluded from the statistical
analyses because fewer than 100 round spermatids
could be scored in their "round-cell" preparations
(8 subfertile men and 1 fertile man). Table 1
shows the mean values of the 3 groups of men
studied: the fertile nonsmokers, subfertile nonsmokers and subfertile smokers. The fertile individuals had significantly fewer abnormal sperm
than the subfertile men. The smokers did not have

TABLE 1
SOME VALUES AND RESULTS OF SEMEN ANALYSES
OF THE MEN STUDIED (mean -+S.D., range)
Fertile

Subfertile

Nonsmokers Nonsmokers Smokers

Number of men
Age(years)
Duration of
abstinence
(days)
Semen volume
(ml)
Sperm density
(millions/ml)
Total sperm
count
(millions)
Abnormal sperm

5
30.2-+5.4
(22-36)

34
31.8_+5.7
(23-56)

'20
31.7_+6.9
(21-47)

4.2_+1.5
(2-6)
3.1+1.4 a
(1.5-5.2)
98.4_+61.2
(31.0-179.0)

4.8+2.5
(1-15)
4.8-+1.7
(1.9-9.0)
65.8+59.1
(0.5-241.0)

5.1-+1.9
(3-10)
3.9_+ 1.5 b
(2.2-7.5)
87.8-+85.3
(0.5-368.0)

295.5_+268.4
(2.5-890.0)
63.2_+16.9
(18-94)
43.7+16.3
(5-65)
0.82-+1.30
(0-5)

347.6_+469.8
(1.0-2024.0)
63.5_+17.3
(27-94)
45.3-+18.4
(0-65)
1.15-+1.42
(0-4)

317.7+221.4
(46.5-627.0)
42.4_+10.0 e
(~)
(27-53)
Motile sperm (%) 56.0+2.2
(55-60)
Micronuclei (%) 1.60-+2.30
(0-5)

a p < 0.05 fertile men compared to nonsmoking subfertile

men.
b p < 0.05 subfertile smokers compared to subfertile nonsmokers.
c p < 0.01 fertile men compared to both groups of subfertile
men.

260
Number
of M N /
100 round

spermatids

5
4
3
2

e..

.e

oe

ee*,

.,b

162o

-~o

Cigarettes smoked/d~y

Fig. 3. Micronucleus frequencies in subfertile men according to

the quantity of cigarettes smoked per day (small dot, 1 individual; large dot, 5 individuals; straight line, mean).

statistically significantly different mean values except for semen volume, which was significantly
lower than among the nonsmoking subfertile men.
In 25 (42%) of the 59 men, round spermatids
containing MN were observed. The mean .frequency of MN for the whole group was 1.00 +
1.41%. 10 individuals (50%) among the 20 subfertile smokers and 13 (38%) among the 34 subfertile nonsmokers had MN in their spermatids.
The frequencies of round spermatids with MN
were 1.15 + 1.39% for the smokers and 0.82 +
1.30% for the nonsmokers. The differences were
not statistically significant. The men were also
grouped according to the number of cigarettes
they smoked per day. Although the group smoking
10-20 cigarettes/day had the highest MN frequency, there was no statistically significant difference compared to the nonsmoking group (Fig.

3).
The length of time smoked was taken into
account in regression analysis by using the variable pack-years. No effect of smoking was detected
(b = 0.015 + 0.018, p > 0.20).
Discussion

This study reports a novel approach to the

study of chromosome damage in human male
germ cells. The results show that spermatids occur
in variable quantities in semen samples and that it
is possible to eliminate 95-100% of spermatozoa

from the sample by a simple centrifugation

technique.
The identification of the germ-cell stages was
based on the descriptions given by Clermont and
Leblond (1955) of human testicular cells. It appears that germ cells which have become detached
from the seminiferous epithelium are n o t in an
optimal environment and the morphologic features of the cell are less clear than in the testis.
Principles of identification of the different cell
types present in the ejaculate are described in this
paper, since no suitable guidelines are available. It
is possible that some of the large round spermatids are diploid, since it has been observed in the
northern vole M i c r o t u s o e c o n o m u s that the
frequency of diploid early spermatids is readily
increased by low doses of radiation (Tates and de
Vogel, 1981; Tates, 1984). In seminal smears, distinguishing immature germ cells from white blood
cells has proved to be difficult (Couture et al.,
1976). However, with the method of fixation described and PAS staining of enriched "round
cells", polymorphonuclear leukocytes are very
easily identified due to their PAS-positive purple
cytoplasm while lymphocytes dearly show a distinct nuclear consistency compared to round
spermatids.
Since round spermatids are rare cells in the
ejaculate and some of them cannot be accurately
classified due to degenerative changes, it was decided that 100 spermatids in the Golgi phase or
cap phase would have to be counted in each slide.
However, 13% of the samples in this study contained too few round spermatids. It was observed
that it is not possible to analyze MN in spermatids
in routine seminal smear preparations unless the
number of immature spermatogenic cells in the
ejaculate is exceptionally high. Seminal smears of
2 men occupationally exposed to irradiation have
been studied by Liming (1985) and the reported
frequencies of micronuclei in immature cells were
3 and 2.3%; however, the details of this study have
not been reported.
The mean frequency of MN for all 59 men was
1.00 + 1.41%, while in the testis of rodents the
frequency of MN is reported to be only about
0.13% in untreated animals (LS.hdetie and Parvinen, 1981; Tates et al., 1983) - - a strikingly
lower frequency. It is possible that damaged

261
spermatids, including those containing MN, are
selectively sloughed from the seminiferous epithelium and are thus present in the ejaculate in
larger quantities than in the testis. However, this is
probably not a major reason for the observed
differences, but instead real differences in the rate
of chromosomal damage between humans and
other mammals may exist. It has been estimated
on the basis of the rate of chromosomal aberrations in human spontaneous abortions and, on the
other hand, of experimental animal data that
chromosomal abnormalities are much more frequent in human gametes than in other mammalian
species. Recently, direct evidence has been obtained by analyzing human sperm chromosome
complements with the aid of the technique of in
vitro penetration of zona-free hamster eggs with
human sperm (Martin et al., 1983; Martin, 1984;
Brandriff et al., 1985).
The reasons for the high frequency of MN in
human ejaculated spermatids were not discovered
in the present study. Cigarette smoking was suspected of being capable of increasing MN frequencies since smoking has been shown to increase chromosomal aberrations in human lymphocytes (Obe and Herha, 1978; Fredga et al.,
1982; Obe et al., 1982; Vijayalaxmi and Evans,
1982). Although micronuclei were more often observed in the samples of smokers than in the
samples of nonsmokers, the difference was not
statistically significant. No dose-response relationship was observed, though the group smoking
10-20 cigarettes/day had the highest mean MN
frequency. This result is in accordance with two
studies on MN in human peripheral blood
lymphocytes which showed no effect of smoking
(Obe et al., 1982; Rudd et al., 1984), although two
smaller studies did show an increase by smoking
(H/Sgstedt et al., 1983a,b). It is known that only a
fraction of all acentric chromosomal fragments
produced by clastogens will end up as micronuclei
in daughter cells, and in lymphocytes MN are not
sensitive enough to pick up differences which can
be seen when analyzing chromosome aberrations
(Obe et al., 1982). Compared to lymphocyte cultures, the number of cells suitable for scoring in
semen samples is smaller and this reduces the
possibility of detecting the effect of an exposure.
There have been conflicting data on the possi-

ble harmful effects of tobacco smoke on human

sperm (Wyrobek et al., 1983). Increased numbers
of morphologically abnormal sperm among
smokers have been observed by several groups
(Viczi~m, 1969; Evans et al., 1981; Spira et al.,
1981) while others have not observed such differences (Godfrey, 1981; Vogt et al., 1984;
Kulikauskas et al., 1985). Neither was an effect of
smoking on the proportion of abnormal sperm
observed in the present study. However, the fertile
men had significantly fewer abnormal sperm than
the subfertile men which is in accordance with
earlier findings (Spira et al., 1981; Rogers et al.,
1983). Semen volume is mainly an indicator of the
function of accessory sex glands. The lower semen
volume of smokers compared to nonsmoking subfertile men observed in the present study is probably not due to smoking since nonsmoking fertile
subjects had even smaller semen volumes (Table
1). No effect of smoking on semen volume has
been observed in larger studies (Spira et al., 1981;
Vogt et al., 1984).
The men participating in this study reported
their occupational exposures, previous diseases,
alcohol consumption and therapeutic drugs on the
questionnaires. These factors may have affected
the results obtained, but due to the variety of
different factors and small number of cases, the
effect of any particular exposure, disease or drug
could not be evaluated.
In conclusion, this study is the first attempt to
apply the micronucleus method to human male
germ ceils present in the seminal fluid. Genotoxic
exposures such as tobacco smoke might influence
the frequency of MN in human spermatids but no
effect was observed in this study among subfertile
men. Regarding the relatively high incidence of
MN among the 5 nonsmoking fertile donors, it is
improbable that the result would have been different if only selected normal men had been studied.
A larger study among normal semen donors would
help to clarify the background level of MN and to
evaluate the influence of environmental exposures,
especially if the number of round spermatids
scored could be increased by further development
of the method.
This study also shows that among subfertile
men the frequency of chromosome damage in
germ cells, as observed by the amount of micro-

262
n u c l e a t e d s p e r m a t i d s in s e m i n a l fluid, is c l e a r l y
h i g h e r t h a n w h a t h a s b e e n o b s e r v e d in the g e r m
cells o f u n e x p o s e d r o d e n t s . W h e t h e r this r e f l e c t s
i n n a t e s p e c i e s - s p e c i f i c d i f f e r e n c e s o r results f r o m
t h e m u l t i t u d e of e n v i r o n m e n t a l a n d o c c u p a t i o n a l
e x p o s u r e s o f h u m a n m a l e s r e m a i n s to b e solved.

Acknowledgments
The assistance of Markku Kallajoki, M.D.,
Andrological Laboratory, Institute of Biomedicine, U n i v e r s i t y o f T u r k u , a n d P e r t t i M u t a n e n ,
M.Sc., D e p a r t m e n t o f E p i d e m i o l o g y , I n s t i t u t e o f
Occupational
H e a l t h , H e l s i n k i , is g r a t e f u l l y
a c k n o w l e d g e d . Prof. M a r j a S o r s a is a c k n o w l e d g e d
for h e l p f u l s u g g e s t i o n s r e g a r d i n g t h e m a n u s c r i p t .

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