/ . Embryo/, exp. Morph. Vol. 25, 2, pp. 223-236, 1971
Printed in Great Britain
223
Hop-sterile, a mutant gene affecting sperm
tail development in the mouse
By D. R. JOHNSON 1 AND D. M. HUNT 1
From the M.R.C. Experimental Genetics Unit, Department of Animal
Genetics, University College London
SUMMARY
Hop-sterile (hop) is a new mutation in the mouse, the effects of which include complete
male sterility, a 'hopping' gait and preaxial polydactyly of all feet. Ultrastructural studies of
spermiogenesis show that sperm tails are absent or highly modified. Second meiotic division
is frequently abnormal or incomplete, often with four centrioles per cell. These centrioles
usually fail to form fiagella and sperm tail development is arrested.
INTRODUCTION
Although much attention has been directed both to the problem of male
sterility and to the morphology of normal sperm, situations where sterility
results from abnormal sperm production have been largely neglected. The
mutant gene hop-sterile (hop) in the mouse, in addition to preaxial polydactyly
of all four feet and a characteristic 'hopping' gait, leads to complete male
sterility when homozygous, whereas the female is fully fertile. This paper will
be concerned with an ultrastructural study of spermiogenesis in these mice
together with a consideration of the gross morphological effects of the gene.
ORIGIN AND GENETICS
Hop-sterile (hop) arose in an unirradiated stock in March 1967 at the M.R.C.
Radiobiological Unit, Harwell. It was offered to U.C.L. in October 1969.
Segregation data (Table 1) shows that it behaves as a fully penetrant Mendelian
recessive.
MATERIAL AND METHODS
Alizarin red-S preparations were used to study the anatomy of the axial and
appendicular skeleton. Spermiogenesis was examined in mice of various ages
(Table 2). The left testis and its associated ducts was fixed by immersion in
Zenker's fluid and subsequently paraffin-embedded, sectioned at 8 [i and stained
with haematoxylin and eosin, while the right testis was prepared for electron
microscopy. The testis and cauda epidydimis were fragmented in cacodylate1
Authors' address: Department of Animal Genetics, University College London, Wolfson
House, 4 Stephenson Way, London, NWJ 2 HE, U.K.
224
D. R. JOHNSON AND D. M. HUNT
sucrose buffer (pH 7-2), fixed in cacodylate-buffered glutaraldehyde (5 %),
postfixed in osmic acid, dehydrated in ethanols and embedded in Araldite.
Ultrathin sections were obtained with an LKB ultratome, stained with aqueous
uranyl acetate and lead citrate and examined with an AEI EM 6B electron
microscope.
Table 1. Segregation data for hop-sterile
Mating type
No. of
litters
Mean
litter
size
Normal
Polydactylous
P*
+ /hopx+/hop
+ /hopx hop/hop
16
1
5-6
5-7
70
24
20
16
O-75-O-5Of
0-25-010S
* Probability (P) of Xs value exceeding calculated value.
t Tested against 3:1 ratio.
X Tested against 1 :1 ratio.
Table 2. Number of mice examined
No. of mice
Alizarin preparations
Testes examined
Age (weeks)
+ /hop
hop/hop
+ /hop
hop/hop
4
8
12
16
1
3
2
2
4
2
2
3
3
2
2
6
2
3
RESULTS
1. Gross anatomical observations
From the time when they begin to walk, male and female hop homozygotes
exhibit a characteristic 'hopping' gait, using the hind legs simultaneously.
Posture is abnormal, the hind legs being held rather widely spread. Swimming
is defective, the hindquarters being twisted so that first one then the other
hind leg is uppermost. Sinuous tail movements, characteristic of normal mouse
swimming, are absent. Alizarin preparations show that both fore- and hind-feet
have preaxial polydactyly (Fig. 1). In one hop/hop there was frank dislocation of
the hip, and in two others fusion of the manubrium to the adjacent sternebra.
It is unlikely that the 'hopping' derives from the polydactyly, but the involvement of the hip joint may warrant further investigation.
Despite the complete male sterility, dissection of four hop/hop males aged
12 weeks and three normal litter-mates revealed no gross abnormality of the
reproductive system although hop I hop testes were somewhat smaller than normal.
Hop-sterile mutant in mouse
225
The size and arrangement of the seminiferous tubules is normal (Fig. 2) but
there is a complete absence of tailed sperm in the tubule lumina. The spermatogonial nuclei in hop testes appear condensed; in contrast, many of the underlying spermatocyte nuclei are large and appear to be arrested in the early stages
of meiosis.
2. Ultrastructural aspects of spermiogenesis
The following account of normal spermiogenesis is based on observations from
normal control animals and on the description given by Fawcett & Phillips
Fig. 1
Fig. 2
Fig. 1. Left forefoot (A) and hind foot (B) of a. hopjhop male aged 8 weeks. Camera
lucida drawings of alizarin-red clearance preparations. Note preaxial polydactyly.
Fig. 2. Spermatic tubule of a normal 12-week-old mouse (A) and its hop/hop littermate (B). Note absence of sperm tails in (B). x 240.
226
D. R. JOHNSON AND D. M. HUNT
(1969). Before entering meiosis, the type B spermatogonium undergoes a mitotic
division. Cytokinesis at this division and at the following meiotic divisions is
incomplete, the resulting haploid spermatids remaining in cytoplasmic communication via well-defined intercellular bridges. The paired centrioles migrate
from the Golgi region to the opposite pole of the cell, where the distal centriole
takes up a position perpendicular to, and just beneath, the cell membrane. It is
from this structure that the flagellum develops. The proximal centriole is orientated at an angle of 90° to the distal centriole (Fig. 3 A). A thin sheet of dense
material is formed over the perinuclear surface of the proximal centriole - the
Fig. 3. Diagrammatic representation of the normal process of flagellum formation in
the mouse sperm. (A) Early stage. The centrioles (CP, CD) have migrated to the
cell membrane, where a flagellum (F) is being formed. The articular facet (Art)
has just arisen. The Golgi apparatus (G) andthechromatoid body (C) are at the other
pole of the nucleus (JV).
(B) Later stage. The centrioles have moved inwards towards the nucleus, taking with
them the base of the flagellum. The annulus (A) is a specialization of the reflexed part
of the cell membrane. The Golgi apparatus is migrating from the acrosomal region
(Ac) towards the tail end of the cell.
(C) Later stage. The nucleus is elongating and condensed, partly surrounded by the
acrosome. The articular facet of the proximal centriole lies at the implantation fossa
(/) of the nucleus. The remains of the chromatoid body surround the annulus. The
midpiece (M) is forming. Mitochondria (Mt) which will form the tail sheath are
congregating. The manchette (Ma) has formed attached to the nuclear ring (/?).
Fig. 4. (A) Low-power electron micrograph of early hop/hop spermatid; note the two
closely apposed nuclei (Nl5 N2) in a common cytoplasm; x 6500. (B) A bifid spermatid from a hop Ihop $\ note the two proacrosomes (Ac); x 30000.
Fig. 5. Low-power micrograph of a hopjhop spermatid. The centrioles (CP, CD)
are in early stages of flagellum production although still close to the Golgi apparatus
(G). x 10000.
Hop-sterile mutant in mouse
Fig. 4
Fig. 5
227
228
D. R. JOHNSON AND D. M. H U N T
Anlage of the articular surface of the future connecting piece. During this
developmental stage the Golgi/nuclear association forms the pro-acrosomal
complex. The paired centrioles and the base of the flagellum now move towards
the nucleus, drawing with them a fold of cell wall (Fig. 3 B), the proximal member
finally coming to lie in the implantation fossa of the nucleus (Fig. 3C). The
striated midpiece develops and the distal centriole ultimately loses its identity.
-• • ' *,^ .-..* ^ * a
Fig. 6. Two hop/hop spermatids joined by a normal intercellular bridge (arrowed).
Note the complex of four centrioles between the nuclei, x 15000.
The dense outer flagellar fibres which surround the 9 + 2 inner core of the sperm
tail are thought to arise as lateral outgrowths from the wall of the original
flagellar doublets, slender at first but increasing in diameter (Phillips, 1970),
while around this fibrous core mitochondria become organized into the characteristic spiral.
This process in hop homozygotes shows a number of anomalies. Cytokinesis
is frequently interrupted at an early stage, so that two apposed nuclei are contained within a common cytoplasm (Fig. 4 A). Bifid nuclei are also seen (Fig. 4B).
The centrioles may fail to migrate to the opposite pole of the cell from the Golgi
Hop-sterile mutant in mouse
229
apparatus (Fig. 5), and occasionally a well-defined cytoplasmic bridge is seen
containing a complex of four centrioles (Fig. 6). A flagellum may be formed
(Fig. 7C) but it is frequently abnormal, with either a central 9 + 2 configuration
plus an additional element (Fig. 7A) or an 8 + 2 with an outsider (Fig. 7B). In
many cases two pairs of centrioles can be observed within a single cell (Fig. 8 A)
and occasionally both produce a flagellum (Fig. 8B). Both pairs of centrioles
may become attached to a single nucleus in adjacent implantation fossae
B
Fig. 7. Sperm tail fiagella formed by hop/hop mice. (A) 8 + 2 doublets plus an
outsider (arrowed), x 105000. (B) 9 + 2 doublets plus an outsider (arrowed),
x 80000. (A) Through the end piece of the sperm; (B) through the end of the
principal piece. (C) Apparently normal flagellar formation at the cell membrane
of a hop/hop spermatid; the plane of section has missed the centrioles; x 24700.
(Fig. 8C, D). In extreme cases, two sets of centrioles appear to form a single
complex (Fig. 9). The flagellum at this stage may appear almost normal
(Fig. 10 B) or consist only of the central pair of fibres (Fig. 10C). But frequently
it is entirely absent, the centrioles returning to the nucleus with an empty fold
of cytoplasm (Fig. 10D).
Large numbers of single flagellar fibres with poor orientation are frequently
230
D. R. JOHNSON AND D. M. H U N T
VI
D
Fig. 8. (A) Two pairs of centrioles in a hop/hop spermatid before migration to the
nuclear membrane, x 24500.
(B) A similar stage to (C) but with two flagella being formed, x 55000. (C, D) Later
stages of spermatids with two pairs of centrioles. In (C) the upper pair (cut
obliquely) has implanted in the nucleus, the lower pair has not. x 35300. In (D)
both sets of centrioles have implanted in adjacent fossae, x 21400.
Hop-sterile mutant in mouse
231
Fig. 9. Section through a late hop/hop spermatid. There are two masses of nuclear
material (Nly N2) though it is not clear if these are separate or parts of a bifid structure
as in Fig. 4B. Two sets of centrioles seem to have formed a single complex, x 37700.
Fig. 10. (A) Details of flagellar formation in hop/hop normal litter-mate. Note paired
centrioles (CP, CD) forming midpiece (M), flagellum, annulus (A); x 34500. (B, C,
D) hop/hop. (B) Nearly normal but infold of cytoplasm (F) is very wide; x 30000.
(C) Poor flagellar structure, apparently only central two fibres (arrowed) formed;
x 40000. (D) No sign of flagellum; centrioles have returned to the nucleus bringing
only an empty fold of cytoplasm (F); x 34500.
232
D. R. JOHNSON AND D. M. HUNT
observed and these are occasionally surrounded by a disorganized mitochondrial
spiral (Fig. 11). Towards the centre of the spermatic tubule large bodies can be
found, the final products of spermiogenesis (Fig. 12). Tail fibres, sections of
mitochondrial sheath and large vesicles containing a granular substance of low
electron-density are characteristic of these abortive spermatids, while the sperm
heads are highly irregular in shape and often incorporate a portion of the
manchette (Fig. 13).
Fig. .1.1. (A) Near longitudinal section through hop/hop sperm tail; note the many
poorly orientated fibres; x 7000. (B) Attempted mitochondrial sheath in hop/hop.
x 20000.
DISCUSSION
Clearly no useful comment can be made on the hop-sterile syndrome as such.
The abnormalities described (male sterility, polydactyly and 'hopping') appear
unconnected at the present state of our knowledge. However, male sterility and
limb defects are associated in a number of mouse mutants. In Strong's luxoid
(1st) homozygotes display preaxial polydactyly of all four feet. Although
spermiogenesis is normal no offspring are obtained, possibly through inability
to copulate successfully (Forsthoefel, 1962). Homozygotes for dominant
hemimelia (Dh) are sterile, with short, twisted, oligodactylous hind legs and
Hop-sterile mutant in mouse
233
normal forelegs (Searle, 1964). Death is usually early but sperm have been
observed in mature survivors. Another male sterile mutant - postaxial hemimelia (px), also described by Searle (1964) - possesses an abnormal connexion
of the vas deferens to the distal end of the seminal vesicle, thus preventing the
dispersal of otherwise normal sperm.
' •
Fig. 12. (A) The final product: hop/hop 'sperm' consisting of head, vesicles (V) and
attempted mitochondrial sheath; x98OO. (B) 'Sperm tail' with many disorientated
fibres, and adjacent spermatid with two centriolar complexes, one forming a
flagellum; x 7000.
These mutants bear only a superficial resemblance to hop since, in all three
cases, spermiogenesis is normal. A rather better correlation is obtained with
Green's luxoid (lu) where spermatogonia pass through a normal, then a delayed
cycle of spermatogenesis during the first 6 weeks of life (D. Elkins & P. F.
Forsthoefel, personal communication). Few primary spermatocytes are produced from this second cycle and spermatogonia are totally lacking after this
period. Ultrastructural studies have not so far been undertaken on this mutant
but the sperm produced are grossly normal and tailed.
Only one condition similar to hop has been reported. In a recent paper Smith,
Oura & Zamboni (1970) describe a defect in sperm morphology which occurred
B
234
D. R. JOHNSON AND D. M. H U N T
Fig. 13. (A) Grossly abnormal sperm heads from hop/hop testis; x 8500. (B) Rather less
abnormal sperm head, x 12300. Note manchette material (Ma) included in heads.
Hop-sterile mutant in mouse
235
in a random-bred mouse stock. The abnormality consisted 'mostly of alterations
in the number and arrangement of the elements of the axial filament complex
and dense outer fibres, incompleteness of the fibrous sheath and disorganization
of the mitochondria of the middle piece'. The condition seems to be inherited.
This could be a rather less extreme occurrence of the defect found in hop,
although affected mice are in fact fertile and Smith presents evidence that
abnormal sperm can fertilize eggs in vivo.
The sterility in hop males evidently resides in the failure to develop normal
sperm tails. As flagella elsewhere (for example, in the fallopian tube of hop/hop
females) are normal, the defect is probably associated with the apparent inability
of the sperm centrioles to either initiate or maintain a normal flagellar structure.
Although two-tailed or two-headed sperm are not uncommon in mammalian
testes (Bloom & Fawcett, 1968) the presence of binucleate spermatids and double
flagellar structures in this context must not be overlooked. If defective centriole
function is the primary cause of abnormal flagellar formation, it is plausible that
cell division may also be disrupted. Successful centriolar division with asynchronous or abortive nuclear division would produce an apparently uninucleate
cell with two pairs of centrioles. The occasional bifid nucleus may represent
partial failure in nuclear division after second meiosis, and the attachment of
two distinct flagellar structures to a single nucleus is certainly in favour of this
interpretation. Throughout the duration of this study no more than two nuclei
and two pairs of centrioles were noted in a common cytoplasm, in sharp contrast to the situation in pink-eyed sterile mice (/>6H andp 25H ), where up to seven
spermatid nuclei were observed within a single cell (Hunt & Johnson, 1971), and
failure in cytokinesis is presumably not limited to the second meiotic division.
Whether or not a flagellum is developed, migration of the centrioles from the
cell membrane to the implantation fossa of the nucleus is normal. However,
the outer fibres of the sperm tail are very rarely formed and this is in accord
with Phillips' (1970) view that the outer fibres arise from the inner ones. Instead,
there appears to be an uncontrolled proliferation of single fibres, often in rather
large numbers, with an abortive attempt to form a mitochondrial sheath.
The authors are indebted to Dr M. S. Deol for many helpful suggestions and criticisms,
and to Dr R. M. Bellairs for reading the manuscript: also to Mr A. J. Lee for Figs. 1 and
3, and to Miss G. M. Tyler for technical assistance.
REFERENCES
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W. B. Saunders.
FAWCETT, D. W. & PHILLIPS, D. M. (1969). The fine structure and development of the neck
region of the mammalian spermatozoan. Anat. Rec. 165, 153-184.
FORSTHOEFEL, P. F. (1962). Genetics and manifold effects of Strong's luxoid gene in the mouse,
including its interactions with Green's luxoid and Carter's luxate genes. /. Morph. 110,
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BLOOM,
l6
EM B 25
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D. R. JOHNSON AND D. M. H U N T
D. M. & JOHNSON, D. R. (1971). Spermiogenesis in two pink-eyed sterile mutants in
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spermatozoa. Nature, Lond. 227, 79-80.
HUNT,
{Manuscript received 16 October 1970)