/ . Embryo/, exp. Morph. Vol. 25, 2, pp. 223-236, 1971 2 2 3

Printed in Great Britain

Hop-sterile, a mutant gene affecting spermtail development in the mouse

By D. R. JOHNSON1 AND D. M. HUNT1

From the M.R.C. Experimental Genetics Unit, Department of AnimalGenetics, University College London

SUMMARY

Hop-sterile (hop) is a new mutation in the mouse, the effects of which include completemale sterility, a 'hopping' gait and preaxial polydactyly of all feet. Ultrastructural studies ofspermiogenesis show that sperm tails are absent or highly modified. Second meiotic divisionis frequently abnormal or incomplete, often with four centrioles per cell. These centriolesusually fail to form fiagella and sperm tail development is arrested.

INTRODUCTION

Although much attention has been directed both to the problem of malesterility and to the morphology of normal sperm, situations where sterilityresults from abnormal sperm production have been largely neglected. Themutant gene hop-sterile (hop) in the mouse, in addition to preaxial polydactylyof all four feet and a characteristic 'hopping' gait, leads to complete malesterility when homozygous, whereas the female is fully fertile. This paper willbe concerned with an ultrastructural study of spermiogenesis in these micetogether 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 Mendelianrecessive.

MATERIAL AND METHODS

Alizarin red-S preparations were used to study the anatomy of the axial andappendicular skeleton. Spermiogenesis was examined in mice of various ages(Table 2). The left testis and its associated ducts was fixed by immersion inZenker's fluid and subsequently paraffin-embedded, sectioned at 8 [i and stainedwith haematoxylin and eosin, while the right testis was prepared for electronmicroscopy. The testis and cauda epidydimis were fragmented in cacodylate-

1 Authors' address: Department of Animal Genetics, University College London, WolfsonHouse, 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 aqueousuranyl acetate and lead citrate and examined with an AEI EM 6B electronmicroscope.

Table 1. Segregation data for hop-sterile

MeanNo. of litter Poly-

Mating type litters size Normal dactylous P*

+ /hopx+/hop 16 5-6 70 20 O-75-O-5Of+ /hopx hop/hop 1 5-7 24 16 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)

48

1216

+ /hop

132

hop/hop

242

+ /hop

2332

hop/hop

2623

RESULTS

1. Gross anatomical observations

From the time when they begin to walk, male and female hop homozygotesexhibit a characteristic 'hopping' gait, using the hind legs simultaneously.Posture is abnormal, the hind legs being held rather widely spread. Swimmingis defective, the hindquarters being twisted so that first one then the otherhind leg is uppermost. Sinuous tail movements, characteristic of normal mouseswimming, are absent. Alizarin preparations show that both fore- and hind-feethave preaxial polydactyly (Fig. 1). In one hop/hop there was frank dislocation ofthe 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 involve-ment of the hip joint may warrant further investigation.

Despite the complete male sterility, dissection of four hop/hop males aged12 weeks and three normal litter-mates revealed no gross abnormality of thereproductive system although hop I hop testes were somewhat smaller than normal.

Hop-sterile mutant in mouse 225The size and arrangement of the seminiferous tubules is normal (Fig. 2) butthere is a complete absence of tailed sperm in the tubule lumina. The sperma-togonial nuclei in hop testes appear condensed; in contrast, many of the under-lying spermatocyte nuclei are large and appear to be arrested in the early stagesof meiosis.

2. Ultrastructural aspects of spermiogenesis

The following account of normal spermiogenesis is based on observations fromnormal 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. Cameralucida 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 litter-mate (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 mitoticdivision. Cytokinesis at this division and at the following meiotic divisions isincomplete, the resulting haploid spermatids remaining in cytoplasmic com-munication via well-defined intercellular bridges. The paired centrioles migratefrom the Golgi region to the opposite pole of the cell, where the distal centrioletakes up a position perpendicular to, and just beneath, the cell membrane. It isfrom this structure that the flagellum develops. The proximal centriole is orien-tated at an angle of 90° to the distal centriole (Fig. 3 A). A thin sheet of densematerial is formed over the perinuclear surface of the proximal centriole - the

Fig. 3. Diagrammatic representation of the normal process of flagellum formation inthe mouse sperm. (A) Early stage. The centrioles (CP, CD) have migrated to thecell 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 otherpole of the nucleus (JV).(B) Later stage. The centrioles have moved inwards towards the nucleus, taking withthem the base of the flagellum. The annulus (A) is a specialization of the reflexed partof 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 theacrosome. The articular facet of the proximal centriole lies at the implantation fossa(/) of the nucleus. The remains of the chromatoid body surround the annulus. Themidpiece (M) is forming. Mitochondria (Mt) which will form the tail sheath arecongregating. The manchette (Ma) has formed attached to the nuclear ring (/?).

Fig. 4. (A) Low-power electron micrograph of early hop/hop spermatid; note the twoclosely apposed nuclei (Nl5 N2) in a common cytoplasm; x 6500. (B) A bifid sperma-tid 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 227

Fig. 4

Fig. 5

228 D. R. JOHNSON AND D. M. HUNT

Anlage of the articular surface of the future connecting piece. During thisdevelopmental stage the Golgi/nuclear association forms the pro-acrosomalcomplex. The paired centrioles and the base of the flagellum now move towardsthe nucleus, drawing with them a fold of cell wall (Fig. 3 B), the proximal memberfinally coming to lie in the implantation fossa of the nucleus (Fig. 3C). Thestriated 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 spermtail are thought to arise as lateral outgrowths from the wall of the originalflagellar doublets, slender at first but increasing in diameter (Phillips, 1970),while around this fibrous core mitochondria become organized into the charac-teristic spiral.

This process in hop homozygotes shows a number of anomalies. Cytokinesisis frequently interrupted at an early stage, so that two apposed nuclei are con-tained 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 229apparatus (Fig. 5), and occasionally a well-defined cytoplasmic bridge is seencontaining 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 configurationplus an additional element (Fig. 7A) or an 8 + 2 with an outsider (Fig. 7B). Inmany 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 centriolesmay 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 anoutsider (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 theprincipal piece. (C) Apparently normal flagellar formation at the cell membraneof 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 singlecomplex (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 frequentlyit is entirely absent, the centrioles returning to the nucleus with an empty foldof cytoplasm (Fig. 10D).

Large numbers of single flagellar fibres with poor orientation are frequently

230 D. R. JOHNSON AND D. M. HUNT

VI

D

Fig. 8. (A) Two pairs of centrioles in a hop/hop spermatid before migration to thenuclear membrane, x 24500.(B) A similar stage to (C) but with two flagella being formed, x 55000. (C, D) Laterstages of spermatids with two pairs of centrioles. In (C) the upper pair (cutobliquely) 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 nuclearmaterial (Nly N2) though it is not clear if these are separate or parts of a bifid structureas 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 pairedcentrioles (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 bringingonly 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 mitochondrialspiral (Fig. 11). Towards the centre of the spermatic tubule large bodies can befound, the final products of spermiogenesis (Fig. 12). Tail fibres, sections ofmitochondrial sheath and large vesicles containing a granular substance of lowelectron-density are characteristic of these abortive spermatids, while the spermheads are highly irregular in shape and often incorporate a portion of themanchette (Fig. 13).

Fig. .1.1. (A) Near longitudinal section through hop/hop sperm tail; note the manypoorly 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') appearunconnected at the present state of our knowledge. However, male sterility andlimb defects are associated in a number of mouse mutants. In Strong's luxoid(1st) homozygotes display preaxial polydactyly of all four feet. Althoughspermiogenesis is normal no offspring are obtained, possibly through inabilityto copulate successfully (Forsthoefel, 1962). Homozygotes for dominanthemimelia (Dh) are sterile, with short, twisted, oligodactylous hind legs and

Hop-sterile mutant in mouse 233normal forelegs (Searle, 1964). Death is usually early but sperm have beenobserved in mature survivors. Another male sterile mutant - postaxial hemi-melia (px), also described by Searle (1964) - possesses an abnormal connexionof the vas deferens to the distal end of the seminal vesicle, thus preventing thedispersal of otherwise normal sperm.

' • B

Fig. 12. (A) The final product: hop/hop 'sperm' consisting of head, vesicles (V) andattempted mitochondrial sheath; x98OO. (B) 'Sperm tail' with many disorientatedfibres, and adjacent spermatid with two centriolar complexes, one forming aflagellum; x 7000.

These mutants bear only a superficial resemblance to hop since, in all threecases, spermiogenesis is normal. A rather better correlation is obtained withGreen's luxoid (lu) where spermatogonia pass through a normal, then a delayedcycle of spermatogenesis during the first 6 weeks of life (D. Elkins & P. F.Forsthoefel, personal communication). Few primary spermatocytes are pro-duced from this second cycle and spermatogonia are totally lacking after thisperiod. Ultrastructural studies have not so far been undertaken on this mutantbut 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

234 D. R. JOHNSON AND D. M. HUNT

Fig. 13. (A) Grossly abnormal sperm heads from hop/hop testis; x 8500. (B) Rather lessabnormal 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 alterationsin the number and arrangement of the elements of the axial filament complexand dense outer fibres, incompleteness of the fibrous sheath and disorganizationof 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 thatabnormal sperm can fertilize eggs in vivo.

The sterility in hop males evidently resides in the failure to develop normalsperm tails. As flagella elsewhere (for example, in the fallopian tube of hop/hopfemales) are normal, the defect is probably associated with the apparent inabilityof the sperm centrioles to either initiate or maintain a normal flagellar structure.

Although two-tailed or two-headed sperm are not uncommon in mammaliantestes (Bloom & Fawcett, 1968) the presence of binucleate spermatids and doubleflagellar structures in this context must not be overlooked. If defective centriolefunction is the primary cause of abnormal flagellar formation, it is plausible thatcell division may also be disrupted. Successful centriolar division with asyn-chronous or abortive nuclear division would produce an apparently uninucleatecell with two pairs of centrioles. The occasional bifid nucleus may representpartial failure in nuclear division after second meiosis, and the attachment oftwo distinct flagellar structures to a single nucleus is certainly in favour of thisinterpretation. Throughout the duration of this study no more than two nucleiand two pairs of centrioles were noted in a common cytoplasm, in sharp con-trast to the situation in pink-eyed sterile mice (/>6H andp25H), where up to sevenspermatid nuclei were observed within a single cell (Hunt & Johnson, 1971), andfailure in cytokinesis is presumably not limited to the second meiotic division.

Whether or not a flagellum is developed, migration of the centrioles from thecell 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 accordwith 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 ratherlarge 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 and3, and to Miss G. M. Tyler for technical assistance.

REFERENCES

BLOOM, W. & FAWCETT, D. W. (1968). A Textbook of Histology. 9th ed. Philadelphia:W. B. Saunders.

FAWCETT, D. W. & PHILLIPS, D. M. (1969). The fine structure and development of the neckregion 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. 110,391-420.

l6 EM B 25

236 D. R. JOHNSON AND D. M. HUNT

HUNT, D. M. & JOHNSON, D. R. (1971). Spermiogenesis in two pink-eyed sterile mutants inthe mouse. /. Embryol. exp. Morph. (in press)

PHILLIPS, D. M. (1970). Insect sperm: their structure and morphogenesis. / . Cell Biol. 44,243-277.

SEARLE, A. G. (1964). The genetics and morphology of two 'luxoid' mutants in the housemouse. Genet. Res. 5, 171-197.

SMITH, D. M., OURA, C. & ZAMBONI, L. (1970). Fertilizing ability of structurally abnormalspermatozoa. Nature, Lond. 227, 79-80.

{Manuscript received 16 October 1970)