/ . Embryol. exp. Morph. Vol. 26, 1, pp. 111-121, 1971
\ \ \
Printed in Great Britain
Abnormal spermiogenesis in two pink-eyed
sterile mutants in the mouse
By D. M. HUNT 1 AND D. R. JOHNSON 1
M.R.C. Experimental Genetics Unit, Department of
Animal Genetics, University College London
SUMMARY
Abnormal spermiogenesis is described in two neutron-irradiation induced alleles at the
pink-eye locus in the mouse. Testicular hypotrophy and abnormal spermateliosis are present,
resulting in the formation of giant sperm with abnormal heads and multiple tails. Abnormal
sperm-head shape results from abnormal proacrosome formation and multiplication of tail
number from a failure in cytokinesis.
The pleiotropic effects of the locus on coat colour, fertility, behaviour and growth are
reviewed in conjunction with similar mutants in other rodents: it is suggested that pituitary
dysfunction could explain all aspects of the 'pink-eye' syndrome.
INTRODUCTION
The pink-eye locus in linkage group I of the mouse is known as a long allelic
series from spontaneous mutations and from its use in specific-locus mutation
experiments. The major effect is a dilution of coat colour arising from a large
reduction in eumelanin and a smaller reduction in phaeomelanin. In many
allelic combinations pigmentation of the eye is also reduced (Searle, 1968).
Hollander (Hollander, 1960; Hollander, Bryan & Gowen, 1960) gave a preliminary description of an allele Go-sterile, ps) which, in addition to a dilution
in coat and eye pigmentation, reduced body size, affected dentition and produced irregular sperm-head morphology and male sterility. This paper will be
chiefly concerned with an ultrastructural study of spermiogenesis in two further
male sterile p alleles which are now available.
MATERIAL AND METHODS
The two p-sterile alleles examined (pm and p25H) arose during specific-locus
mutation experiments at the M.R.C. Radiobiology Unit, Harwell. The testes
and associated ducts from 17 mice of various ages (Table 1) were processed for
light and electron microscopy as described previously (Johnson & Hunt, 1971).
Mature sperm were collected by stripping the vasa deferentia into 0-93 % saline
solution. The resulting sperm suspension was examined using interference
contrast microscopy.
1
Authors' address: Department of Animal Genetics, Wolfson House, 4 Stephenson Way,
London NW1 2HE, U.K.
112
D. M. HUNT AND D. R. JOHNSON
OBSERVATIONS
p jp
and p lp^
mice are smaller than their normal litter-mates at birth
and rarely achieve the adult weight of their normal sibs. The coat colour is
lighter than in pjp mice but eye pigmentation is reduced to about the same
degree. They are active, nervous and a little uncoordinated. No malocclusion of
the incisors, abnormal wear patterns or other dental defects were seen in mice
of either genotype.
m
m
25H
H
Table 1. Mice used in the preparation of light- and
electron-microscope sections
Genotype
•>
Age (weeks)
pSHjpOH
4
8
12 +
Total
0
3
2
5
+/+
2
3
2
7
2
1
2
5
Table 2. Segregation o/p 6 H and])25H
Mating type
i5H
+ /p x + /p»#
+ x+
pp
x+
Litter
size
5-651
6-49/
410»
5-20/
P*
nn?
UU
nin
Normal
Pink-eye
Total
Pf
/178
\467
/ 21
1114
47
0
6
0
225
467
27
114
0-3-0-2
0-9-0-8
* Probability of t value exceeding calculated value.
t Probability of x* value exceeding calculated value : tested against 3:1 ratio.
Table 3. Distribution of nuclear number in 184 abnormal
spermatids from a p 2 5 H /p 2 5 H testis
No. of nuclei per spermatid
A
No. of spermatids
2
3
4
5
6
1
8
16
110
12
39
5
9
0
8
1
pm and/?25H are inherited as simple recessives to full colour (Table 2). Despite
the good fit to the expected 3:1 ratio from intercross matings litter size was
reduced, significantly so in the larger data from/?257*.
The effects of/?6H and/?2571 on spermiogenesis are identical and no distinction
will be made between them in the text. Illustrations have been selected from both
alleles and are appropriately labelled.
Spermiogenesis in the mouse
113
The testes of/^-sterile mice are less than half the size of those of normal littermates. The spermatic tubules are smaller in diameter (Figs. 1, 2) and the number
of spermatogonia and sperm (as judged by the number of sperm tails visible in
the lumina of the spermatic tubules) is reduced (Figs. 3, 4). Giant rounded
spermatids containing many nuclei are present in small numbers in the germinal
epithelium while spermatids with 16 or fewer nuclei are more common (Table 3).
Figs. 1, 2. Median T.S. through testis of a normal mouse (Fig. 1) and \tsp25Hlp25H
litter-mate. Note the reduced size of the latter.
Figs. 3, 4. Representative spermatic tubules of normal mouse (Fig. 3) a.ndp25H/p25H
litter-mate. Note multinucleate spermatid (arrowed).
Under the electron microscope the development of the acrosome is frequently
seen to be abnormal irrespective of the number of nuclei present in a spermatid.
In some cases the proacrosome is duplicated (Figs. 5, 6). Frequently the proacrosomal vesicle and underlying nuclear membrane become considerably
8
EMB 26
114
D. M. HUNT AND D. R. JOHNSON
Spermiogenesis in the mouse
115
flattened (Fig. 11) or irregular in outline (Fig. 12) and, at a later stage, vesicular
inclusions can be seen in the developing acrosome (Fig. 7).
Many spermatids contain two or more nuclei (Figs. 11,12) and in some of these
a single Golgi complex is seen equidistant from two nuclei (Fig. 5) or closely
apposed Golgi regions appear to be in the process of fusion (Fig. 8). At a later
stage acrosomal material is laid down in the narrow spaces between adjacent
nuclei (Fig. 9).
Maturing sperm from the lumen of the spermatic tubule and from the cauda
epididymidis show various abnormalities of head morphology (Fig. 10); cf.
Hollander et ai (1960). In the vasa deferentia a number of giant sperm characterized by grossly altered head shape and a multiplicity of tails are present
(Figs. 13-15). In e.m. sections of the cauda epididymidis up to seven tail
flagella have been identified within a single cell membrane (Figs. 16-21), often
apparently cut at different levels. The mitochondrial spiral characteristic of
normal sperm tails fails to develop or is poorly organized.
DISCUSSION
Sterility in male mammals, whether' spontaneous' (i.e. of unknown aetiology),
genetic or induced, usually arises from germinal hypoplasia, meiotic upset or
abnormal spermateliosis. These categories are not necessarily mutually exclusive. Hypoplasia is well known in man, laboratory and domestic animals,
and meiotic upset has often been described, especially in interspecific hybrids,
but cases of abnormal spermateliosis are infrequently reported.
The small size of the testes and seminiferous tubules together with the reduced
number of spermatogonia in pm and p25H point to germinal hypoplasia. The
development of those spermatocytes which remain is clearly abnormal. Hollander
et al (1960) suggested, without supporting evidence, that irregular sperm-head
morphology in/?s-mice resulted from abnormal acrosome development, and the
results presented here confirm this assumption. Acrosome development is indeed
abnormal in the two p alleles studied, even in the quasi-normal situation of one
nucleus per spermatid. Superimposed upon this are abnormalities of acrosome
formation apparently resulting from the close proximity of two or more nuclei.
In such cases acrosomal material may unite several nuclei. It is open to question
Fig. 5. Binucleate spermatid from p25H. Note the double proacrosome (arrowed)
and Golgi region (G) equidistant from the two nuclei (NUN2.)
Fig. 6. Double proacrosome (arrowed) from a uninucleate p25H spermatid.
Fig. 7. Later stage of proacrosome formation showing vacuolation. p25H.
Fig. 8. Binucleate spermatid fromi?6^ showing two adjacent Golgi regions (d,G 2 ).
Fig. 9. Group of 4 nuclei in a pi5H spermatid. Note disposition of electron-dense
proacrosome material in interstices.
Fig. .10. Abnormal sperm heads inpGH germinal epithelium.
8-2
116
D. M. H U N T AND D . R . J O H N S O N
Fig. 11. Survey of part of p25H seminiferous epithelium showing binucleate spermatids (upper right, lower left), one with flattened proacrosomal region (arrowed).
Fig. 12. Multinucleate pm spermatid showing parts of five nuclei (./VWV5) and
abnormal proacrosome (arrowed).
Spermiogenesis in the mouse
Figs. 13—15. Interference-contrast micrographs of abnormal sperm from the
vasa deferentia of pm mice.
Figs. 16-21. Sections through tail regions of giant sperm from the epididymis of
pMH Note multiple flagella and disorganized mitochondrial spirals (M).
117
118
D. M. HUNT AND D. R. JOHNSON
whether this common acrosome structure would occur in a spontaneous multinucleate spermatid such as is occasionally found in laboratory rodents. We
suspect that it would not. Parkes (1960) reproduces a phase-contrast micrograph
of two spontaneously occurring multinucleate guinea-pig spermatids, one containing eight, the other 16 nuclei. The characteristic guinea-pig acrosome is
visible on almost all nuclei with no signs of fusion.
The presence of spermatids with various numbers of nuclei (Table 3) argues
against any stage specific effect on cytokinesis. This is in contrast to the situation
in hop-sterile (Johnson & Hunt, 1971), where spermatids contain one or two
nuclei only, and failure in cytokinesis can be ascribed to the second meiotic
division. The fact that spermatids are normally interconnected by cytoplasmic
bridges (Burgos & Fawcett, 1955; Fawcett, Ito & Slautterback, 1959) and that
'the first and most pronounced result of spermatogenic damage is generally
arrest of spermateliosis with exfoliation of spermatids and the appearance of
round multinucleate cells' (Parkes, 1960) leads us to suppose that failure of
cytokinesis here is secondary to other derangements of the germinal epithelium.
Sperm tail development is relatively unimpeded, with each pair of centrioles
producing a well-organized 9 + 2 flagellum. In transverse sections through the
flagella of giant sperm the size and disposition of the outer fibres indicate
section at apparently different levels. This seems unlikely as the nuclei, and
hence the centrioles giving rise to the flagella, are adjacent. Phillips (1970) has
shown that the outer tail fibres are formed from the 9 + 2 inner core, and it may
be that this process is suppressed by the abnormal conditions present within the
giant sperm.
Abnormal acrosome formation leading to sterility has also been described in
some of the t alleles of the mouse (Bryson, 1943; Rajasekarasetty, 1954; Braden
& Gluecksohn-Waelsch, 1958; Johnston, 1968) and in Friesian bulls (Hancock
& Trevan, 1957), where the condition has been shown to be inherited (Donald &
Hancock, 1953). In the present case, however, sterility is associated with other
pleiotropic effects, primarily in pigment synthesis.
Two well-documented and distinct syndromes affecting both pigmentation
and fertility exist in mammals, the 'white' syndrome and the 'pink-eye' syndrome. The 'white' syndrome is characterized by little pigment in the coat and
variably pigmented eyes. Sterility and anaemia are also commonly present.
Examples given by Parkes (1960) and Searle (1968) include the W and SI loci
in mouse, Swedish Highland cattle, Hedlund white mink, and merle in the dog.
In the Wmouse (and in other species by implication), sterility depends on the
failure of the primary germ cells to reach the gonads, or their arrival in very
reduced numbers. Both sexes are affected equally. The pigment defect is considered as a failure in melanoblast migration, which, in the mouse, occurs
concurrently with primary germ-cell migration (Mintz, 1960). Abnormal
spermateliosis also occurs in some cases (Swedish Highland cattle (Parkes, 1960);
Wj+ and W°l+ mice (Hunt & Johnson, unpublished observations)).
Spermiogenesis in the mouse
119
The second syndrome, 'pink-eye', characterized by a reduction in the
eumelanin of the coat and reduced pigmentation of the choroid of the eye, is
known only in rodents: mouse (Hollander et al. 1960, and this paper); golden
hamster (Robinson, 1955, 1958; Bruce 1958); Mastomys natalensis (Menzies,
1957) and guinea-pig (Jakway & Young, 1958). The associated male sterility arises
from testicular hypoplasia, abnormal spermatogenesis and abnormal spermateliosis.
At the cellular level the common factor in spermatocyte and melanocyte
development is the Golgi apparatus. The role of this cell organelle in spermateliosis is undeniable (see Dan, 1970, for a review) and involvement in melanosome formation in the melanocytes of the skin is well documented, although
there is no evidence for participation of the Golgi region in melanosome development in the mouse retina (Foster, 1965).
The 'pink-eye' syndrome as a whole, however, involves more than acrosome
formation and coat colour, and the following abnormalities have also been
reported in some or all of the known 'pink-eye' mutants (Table 4).
Table 4. Pleiotropic symptoms in the 'pink-eye' syndrome
Diluted
coat
colour
3
sterility
9
fecundity
affected
Reduced Abnormal
growth behaviour
Mouse
ps
+
+
+
+
+
Hamster
Guinea-pig
Mastomys
ru
p
.
+
+
+
+
+
+
+
—
—
+
—
.
+
—
.
+ , present; —, absent.
(1) Body size is reduced in the ruby hamster (Robinson, 1958), p8 (Hollander,
1960; Hollander et al. 1960) and/?67* and/?251*. Even/? homozygotes, although
perfectly fertile, 'tend to be smaller than normal litter-mates' (Searle, 1968).
(2) jf homozygotes are active but somewhat uncoordinated (Hollander et al.
1960). Likewise pm and p25H mice show abnormal behaviour and ruby hamsters
are nervous, easily startled and excitable (Robinson, 1958).
(3) Reduced female fecundity has been noted in ps mice (Hollander et al.
1960) and in the ruby hamster (Robinson, 1955).
The pituitary hormones are known to control fertility, coat colour and
moulting, growth and behaviour. The hypophysectomized rat has the following
characteristics (Turner, 1966): cessation of growth, suppression of moulting,
atrophy of the adrenal cortex, atrophy of the thyroid, dysfunction of the testis
and ovary. Panhypopituitarism in man (Sayers, 1954) is a consequence of a
deficiency of all the trophic hormones of the adenohypophysis. Its classic
120
D. M. HUNT AND D. R. JOHNSON
symptoms are similar to those of the hypophysectomized rat. In addition, loss
of pigmentation is a common feature.
The pituitary is intimately involved in the pigmentation of the lower vertebrates; it is implicated in mammals only in disease or in cases of changed winter
pigmentation. Some species of mammals have a lighter hair colour in winter
(Bissonette & Bailey, 1944, describe colour change in the New York weasel
where the dark brown summer coat gives way to a winter coat which is white
or a 'lighter brown or yellowish tinge') and the change is mediated via the
effect of a changing day-length on the hypophysis.
We suggest that the pleiotropic effects of the 'pink-eye' syndrome can be
explained on the basis of a pituitary dysfunction, and we hope to submit further
evidence to test this hypothesis in due course.
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{Received 1 December 1970)