J. Cell Sci. 6, 177-194 (i97o) 177Printed in Great Britain

DEVELOPMENT, ORGANIZATION, AND

DEGENERATION OF THE DROSOPHILA

SPERM FLAGELLUM

B. I. KIEFER

Department of Biology, Wesleyan University, Middlctoiun, Connecticut, U.S.A.

SUMMARY

The development and degeneration of sperm from Y-mutant, sterile Drosophila melanogasterwere studied with emphasis on the differences among the various flagellar microtubules and thephysical linkages among the components of the axial fibre complex.

During the degenerative process the axoneme is disrupted by the breaking of connexionsbetween adjacent peripheral groups (doublet + satellite) and between the secondary and centralfibres, and the microtubules are lost in a definite sequence: (1) inner portion of B-tubule,(2) outer portion of A-tubule, (3) inner portion of A-tubule, (4) outer portion of B-tubule,(5) central and accessory tubules. The observations support the conclusion that the variousmicrotubules are compositionally different and, further, that the A- and B-tubules are, in turn,composed of differing outer and inner halves.

Observations on the development and subsequent breaking apart of the various flagellarstructures demonstrate the presence of the following physical linkages: (1) satellite-to-satellite,(2) A-tubule to adjacent B-tubule, (3) A-tubule to adjacent satellite (accessory tubule), (4)A-tubule to secondary fibres, (5) secondary fibres to central tubules, and (6) a connexion be-tween the two central fibres.

INTRODUCTION

During the course of a study of the ultrastructural defects associated with spermio-genesis in various Y-mutant stocks of Drosophila melanogaster, it was observed thatmasses of sperm were present in various stages of degeneration (Kiefer, 1968). Thedegeneration of the axial fibre complex was seen to progress in a regular sequence ofevents which are relevant to the question of structural similarity among various kindsof sperm microtubules. Additionally both the manner in which the axial fibre complexbreaks up and the development of certain types of incomplete axial fibre complexes inthese mutant flies enable one to examine critically the relationship between thevarious components of the axoneme.

Although the ultrastructure of insect sperm has been the subject of numerousreports (for example, Andre, 1961; Bawa, 1964; Kaye. 1964; Phillips, 1966; Cameron,1965), many of the details of the structure and organization of the axial fibre complexremain unclear. Of particular interest is the possibility of interconnexions among thevarious components of the axial fibre complex. Allen (1968) has recently described afilament connecting adjacent peripheral doublets in Tetrahymena cilia. The 'spokes'(Afzelius, 1959) seen in most flagella and cilia are thought to link the doublets with thesecondary fibres and, in some cases (e.g., Gibbons, 1963), the secondary and central

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178 B. I. Kiefer

fibres, although the latter connexion is obscure in most published pictures. At bestit has been difficult to distinguish between the close proximity of flagellar structuresand their actual physical connexion.

Closely allied to or as a part of studies on axoneme morphology has been a con-sideration of the substructure and function of all cellular structures termed ' micro-tubules', such as the characteristic '9 + 2' tubules of cilia and flagella, the spindlemicrotubules, and those which have been lumped together as 'cytoplasmic micro-tubules'. One of the more pressing questions is whether all microtubules are the same.Although differences in size, electron density, and fixation properties have been noted(see Behnke & Forer, 1967, for review), the striking similarity of the substructure of allmicrotubules examined by negative-staining techniques has led to the postulation of abasic equivalence of all microtubules. Behnke & Forer (1967) have concluded thatmicrotubules fall into 4 classes on the basis of responses to various experimentaltreatments such as temperature changes, fixation procedures, and pepsin digestion.

The present paper deals specifically with the development and subsequent loss ofthe various components of the axial fibre complex of the Drosophila sperm flagellum.The observations presented lead to the conclusions that all members of the axial fibrecomplex are interconnected, and that there exist several classes of compositionallydifferent flagellar structures. A general account of spermiogenesis and the gross aspectsof sperm degeneration in the Y-mutant flies has been presented elsewhere (Kiefer,1968, 1969).

MATERIALS AND METHODS

Drosophila stocksThe sterile Y-tester stocks used in this study have been described in detail by Brosseau (i960).

Briefly, specific deletions in the Y chromosome are maintained in stocks in which the femaleshave attached-X chromosomes and a free sterile Y, and the males have the long arm of the Yattached to the X and a free sterile Y. Sterile males are obtained by crossing stock males withvirgin wild-type females. The flies were grown on standard cornmeal-molasses-agar medium at25 °C. Upon emergence, the males were collected at 12-h intervals and aged for 4 days.

Electron microscopyTestes from 4- to 5-day-old males were dissected in Beadle-Ephrussi saline and fixed for

30 min in cold 6 % glutaraldehyde in 01 M cacodylate buffer (pH 7-3) and post-fixed for 1 h incold 1 % OsO4 in veronal-acetate buffer (pH vs). After dehydration in a series of cold ethanols,the testes were treated with propylene oxide and embedded in Epon 812. Thin sections wereobtained using a Porter-Blum MT-2 ultramicrotome with a glass knife, stained with uranylacetate or lead citrate, and examined in a Zeiss EM—9A electron microscope.

The observations to be reported here are based on a study of random sections of at least10 testes from each of 8 genotypes. In all, well over a thousand sections were examined.

OBSERVATIONS

Development of the axial fibre complex

Partial descriptions of the structure and development of the axial fibre complex ofD. melanogaster have been given previously (Daems, Persijn & Tates, 1963; Biarati,1967; Kiefer, 1966; Meyer, 1968). In the following account emphasis is placed on those

Drosophila sperm flagellum 179

points not specifically dealt with in the above papers. A typical cross-section throughthe main piece of a fully mature D. melanogaster sperm is shown in Fig. 1. Unlike thesperm of many animal species, Drosophila sperm are extremely long (i-8 mm) and donot appear to be structurally differentiated along their length (Meyer, 1968; Biarati,1967; Kiefer, 1966) except for the head region and the very tip of the tail. Hence,Fig. 1 is representative of a section taken almost anywhere along the length of themature sperm. The two major components which comprise the mature flagellum arethe dense mitochondrial element (Nebenkern derivative), and the axial fibre complex.In addition to the typical ' 9 + 2' organization of flagellar microtubules, the axial fibrecomplex contains a peripheral group of 9 paired 'satellites' (Daems et al. 1963) and9 pairs of secondary fibres between the outer and central microtubules.

The axial fibre complex develops from the centriole which lies in a fold of thenuclear membrane at the base of the nucleus (Fig. 2). Adjacent to the centriole theflagellum appears as a simple ring of 9 doublets (Fig. 3). There are projections fromthe B subfibres which are presumed to be continuous with the third member of theperipheral groups seen in the centriole. These projections have been reported in otherinsect sperm (Cameron, 1965; Behnke & Forer, 1967) and have been mistakenly re-ferred to by Meyer (1968) as the classical 'arms' associated with the A subfibres. Thiserror has lead to an inaccurate description of axoneme morphogenesis in Drosophila(Meyer, 1968; Hess & Meyer, 1968). The arms are not present at this stage of develop-ment nor are the central fibres, secondary fibres, spokes, or satellites. As the flagellumlengthens the central fibres and spokes appear, although the latter are not yet clearlydefined. The temporal sequence of flagellar development accompanying spermatidelongation is as follows. The arms become visible and the projections from the B sub-fibres give rise to a third set of tubules (accessory tubules, Figs. 4-6). An area of in-creased electron density appears adjacent to the accessory tubules and together theyform the 'satellites' (Daems et al. 1963). The non-tubular nature of the denser portionof the satellite has been described by Cameron (1965). The accessory tubules alsodevelop a projection which becomes more clearly defined in later stages (Figs. 6, 7). Bythe time all the accessory tubules have been formed, the secondary fibres are alsopresent and there is an indication of some electron-dense material between the secon-dary and the central fibres. After this stage, the developmental changes consist of in-creased density of the satellites, the spokes and secondary fibres, and finally, the centralpair (Figs. 6, 7). In the final stages of development the accessory tubules and thecentral tubules develop an electron-dense 'fibre' in their centres (Fig. 7).

Degeneration of the axial fibre complex

Those sperm which have reached the maximum extent of development typical of theparticular mutant undergo degeneration. Thus, sperm which begin degenerating atsomewhat different stages of development can be observed. The range is from fullymature sperm to those which proceed no further than that typical of males completelylacking a Y chromosome (Kiefer, 1966; Meyer, 1968). In the latter case, developmentis still quite extensive but many of the components of the axial fibre complex have notreached maximum density and can, therefore, be seen better. It should be noted that

180 B. I. Kiefer

the degeneration of the axial fibre complex often takes place with the plasma membraneintact, and that the sequence of breakdown is always the same. The first sign of de-generation is the disorganization of the axial fibre complex. This begins as a separationof adjacent satellites (Fig. 8) and often results in the complete loss of the cylindricalorganization of the axoneme (Fig. 9). As the axoneme loses its organization, the armsof subfibre A and a large portion of subfibre B are lost (Fig. 10). It is difficult to tellwhether these structures are lost simultaneously or whether one closely follows theother. Some sections show axial fibre complexes which appear to have lost all of thearms but only some of the B fibres. However, the arms are normally difficult to see.The loss of the B-fibre component begins at the inner junction of the B and A sub-fibres and progresses toward the accessory fibre, so that all that remains of the B fibreat this stage is the outer half (Fig. 10).

After this stage several components are lost in succession. The sequence appears tobe as follows: spokes and secondary fibres; all but the outer rim of the dense non-tubular member of the satellites and the outer portion of subfibre A; the inner portionof subfibre A; the remaining B-fibre component and the arm of the accessory fibre(Figs. 11, 12). This leaves the two central fibres and the accessory fibres attached to theremaining rim of the non-tubular satellite member (Fig. 13). These fibres are readilyidentified due to the presence of a 'fibre' in their centres. All these remaining com-ponents are then lost simultaneously.

The length of time between successive stages of degeneration can be estimated on thebasis of the frequency with which one sees each of the various stages in any givensection, though this interpretation must be treated with caution. The most commonstages are the first and last (disorganization of the axial fibre complex and only centraland accessory fibres remaining). The second stage (loss of inner component of B fibre)is frequently observed, whereas the remaining stages are only rarely seen.

Structural organization of the axial fibre complex

On the basis of the observations on both developing and degenerating axial fibrecomplexes, some points can be made regarding connexions among the various flagellarcomponents. Figures 9, 14, and 16 demonstrate clearly that the satellites, doublets,spokes, and secondary fibres form an interconnected unit. Even when the axial fibrecomplexes are completely disorganized, either during abnormal development or de-generation, these structures remain together as a unit, and it is the units which arerandomly scattered, not the individual components which comprise them. The con-nexion between the accessory fibre of the satellite and the doublet is obvious fromobservations on the development of the accessory fibre from the B fibre projection. Thephysical linkage of the doublets to the secondary fibres via the spokes has never beenseriously questioned. However, the secondary fibres may not be fibres at all but rathermodifications of the inner end of the spokes. The observations that the secondaryfibres form only after the spokes are well developed (compare Figs. 4-6), and that bothof these structures are digested simultaneously are consistent with this idea. Thespokes are seen in longitudinal sections as alternating structures while visualization ofthe secondary fibres as continuous elements has not been reported.

Drosophila sperm flagellum 181

It may be questioned how a digestive process could produce the kind of disorgani-zation illustrated by Fig. 9. One possibility would be the digestion of a 'matrix' whichholds the axial fibre complex components in a circular array. The validity of this con-cept cannot be argued on the basis of the observations reported here. However in-stead of, or in addition to, such a matrix, the circularity could be maintained if adjacentperipheral units (satellites plus doublets) were connected and/or if the secondaryfibres were connected to the central fibres. A loss of these connexions could result inthe observed disorganization. Among the peripheral units there appear to be con-nexions between adjacent satellites, between adjacent doublets, and between the Afibres and adjacent accessory fibres. The satellite-to-satellite connexion is between theaccessory fibre arm and the non-tubular element of the adjacent satellite. This is mostclearly seen when these units are separating from each other during degeneration(Fig. 8). Often the non-tubular satellite material remains attached to the accessoryfibre arm after adjacent satellites have become completely separated (Fig. 14). Both ofthe A-fibre arms appear to be forked structures, although they are difficult to visualizein detail in any given micrograph. One segment of the inner arm connects an A fibrewith the B fibre of the adjacent doublet, while the outer arm connects to the adjacentaccessory fibre (Figs. 1, 5-8, 15). Connexions between secondary and central fibres canbe seen in most sections of mature sperm (Figs. 1, 7, 8), but are lost during degenera-tion (Figs. 8-14). The two central fibres also appear to be linked by a short fibre (Figs.7. 11). Although this connexion is not always apparent, its existence is supported bythe observation that the two central fibres always remain together after complete dis-ruption of the axial fibre complex (Fig. 16).

The organization of the components of the mature axoneme shown diagrammati-cally in Fig. 17 is based on a study of many micrographs of all stages of developmentand degeneration.

DISCUSSION

Classes of flagellar microtubules

The observations reported here are in excellent agreement with those of Behnke &Forer (1967). Although minor differences exist, the two sets of observations lead to thesame basic conclusion that the various flagellar microtubules are different from eachother and fall into three main classes: (1) A-tubules, (2) B-tubules, and (3) central andaccessory tubules. In addition, it is suggested that the walls of both the A- and the B-tubules are each composed of different materials comprising roughly the outer andinner half of each tubule.

As opposed to the experiments of Behnke & Forer (1967), the present study dealswith a totally in vivo system. The sperm are being digested by normal cellular mecha-nisms. Although the exact nature of these mechanisms is unknown in this system, it isclear that large phagocytic cells are involved, and it seems reasonable to assume thatdegradative enzymes play a major role in the destruction of the sperm. It follows thatseveral different enzymes may be involved, and that their relative concentrations couldvary both temporally and spatially. These considerations strengthen rather than negate

182 B. I. Kiefer

the conclusions drawn from the observations, since all flagellar structures are ultimatelydestroyed and the sequence of destruction is always the same. There are three con-ditions, other than compositional differences, which could lead to a regular, repeatablesequence of fibre loss: (i) the rate of penetration of the 'digestive factors' from theoutside to the centre of a given sperm; (2) variations in the microenvironment of thevarious flagellar microtubules which, in turn, could have differential effects on thedigestive processes; and (3) differences in the material contained within the core of themicrotubules. Of course these possibilities are not mutually exclusive and are probablyall operating. If the flagellar microtubules were identical in composition, and thesequence of loss were due to penetration rate, then one would expect the outermostaccessory tubules to be digested first and the innermost central tubules last. As it turnsout, these two classes are the last to be lost and they are lost simultaneously. Molecularvariations in the environment must indeed exist, but it is doubtful that they could bedetermined in a more meaningful way than in terms of the developmental origin andvisible structural associations of the various microtubules. On these grounds theaccessory and central tubules would appear to be in the most dissimilar environment,yet they are lost simultaneously.

Differences in core material seem to be evident on the basis of the different stainingcharacteristics of the lumens of the various microtubules and the presence of a central'fibre' within the accessory and central tubules (see Behnke & Forer, 1967, for a reviewof various fixation and staining differences among microtubules). This considerationcould lead to exactly the same three classes of flagellar tubules as previously listed.However, the pattern of digestion of the B-tubule could be explained only with greatdifficulty in these terms. It appears then that the observations reported here are bestexplained in terms of compositional differences among various flagellar micro-tubules.

This interpretation must be reconciled with the report of Shelanski & Taylor (1968)on the strikingly similar (if not identical) properties of the protein subunit of the cen-tral and peripheral microtubules of sea-urchin sperm. According to these authors bothproteins have the same sedimentation constants, molecular weights, and RF values ondisk electrophoresis. The slight differences in amino acid composition are within thereproducibility expected for identical samples. Both proteins possess a binding site forguanine nucleotide, although the ability of the isolated proteins to bind guanidinetriphosphate (GTP) was demonstrated only for the central-pair protein. The central-pair protein binds colchicine, while colchicine binding has not been demonstrated forthe outer-doublet protein. These apparent discrepancies have been reasonably ex-plained in terms of experimental procedures causing protein denaturation. However,the possibility that there are real differences in GTP and/or colchicine-binding abilityof the two proteins is not ruled out. As pointed out by Shelanski & Taylor (1968) andRenaud, Rowe & Gibbons (1968), the amino-acid composition of microtubular proteinis strikingly similar to that of actin, yet these two proteins differ in a number of pro-perties, some (but not all) of which could be explained in terms of the configurationalstate of the molecule. Additionally, Shelanski & Taylor (1968) have found that themicrotubular dimer readily loses the colchicine and GTP-binding sites without change

Drosophila sperm flagellum 183

in molecular weight or sedimentation constant, suggesting that the dimer may exist intwo or more configurations.

The ' property' of the flagellar microtubules under consideration in the present paperis the response to cellular digestive mechanisms which most probably include degra-dative enzymes. It is clear from the observations that there is indeed a difference inresponse among the various microtubules. However, these differences could be due toconfigurational differences of identical subunits as well as compositional differences. Ineither case, the point remains that these tubules are different and that these differencesprobably have a functional significance. Finally, it should be noted that insect spermflagella are exceedingly more complex structures than sea-urchin sperm flagella.Although the ' 9 + 2' pattern of flagellar microtubules has been preserved by evolution,it need not follow that compositional identity of microtubules has also been preserved.Hence, sea-urchin sperm microtubules could be identical while those of insects (andother organisms) may simply be evolutionarily related.

Functional organization of flagella

Most of the flagellar structures reported here for Drosophila sperm have been figuredif not described for other insect sperm (Bawa, 1964; Andr6, 1961; Kaye, 1964;Cameron, 1965; Behnke & Forer, 1967; Phillips, 1966). The sub-structure of theflagellar fibres as seen in sectioned material has been discussed in detail by Cameron(1965) and Phillips (1966) and need not be reviewed here again. What has not receivedspecific attention is the presence of structural connexions among various components ofthe axoneme. The present observations support the view that all components of theaxial fibre complex are physically linked. Some of the linkages are obvious only whenthe axoneme begins to break apart. This is particularly true of the satellite-to-satelliteand the satellite-to-doublet connexions.

Although it is premature to present a detailed hypothesis regarding the relation ofthe structural organization of the axoneme to flagellar movement, some general pointscan be made.

In order for a flagellum to perform its characteristic bending movement it needs, atthe very least, elements of contraction, conduction and rigidity (compression). Theseelements must be coordinated in such a way as to provide for a phase difference in thewave of contraction as it passes around the circumference of the axoneme as well asalong its length. Though it is generally argued that the outer doublets are the con-tractile elements, no flagellar fibres can yet be assigned a specific function with anydegree of certainty. It does seem certain that all axoneme structures are necessary forproper flagellar motility, and that the individual elements must interact in a very highlyco-ordinated fashion. Such co-ordination would be facilitated by the interconnexionsdescribed here. As an example of how such a system could work, we may consider thelinkages of the peripheral units around the circumference of the axoneme. Gibbons(1963) has provided compelling evidence that the arms of subfibre A are the sites ofATPase activity which provides the energy for contraction by the splitting of ATP.Daems et al. (1963), using cytochemical techniques on D. melanogaster sperm, con-cluded that the ATPase was located in the 'intersatellite spaces'. A re-examination of

184 B.I. Kiefer

their electron micrographs in the light of the observations presented here shows that thelead phosphate precipitate could be over the outermost A-fibre arm or the accessoryfibre arm, or both. Both of these arms form physical linkages between doublet-accessoryfibre units. The presence of ATPase molecules within these connecting structurescould provide a mechanism for the passage of a wave of contractions around the cir-cumference of the axoneme as well as a physical basis for the sliding model of axonememovement proposed by Satir (1967).

The author wishes to acknowledge the excellent technical assistance of Mrs Jean Bertman andthe support of the USPHS (Grant GM 14726-02).

REFERENCES

AFZELIUS, B. (1959). Electron microscopy of the sperm tail. Results obtained with a newfixative, jf. biophys. biochem. Cytol. 5, 269—278.

ALLEN, R. D. (1968). A reinvestigation of cross-sections of cilia. J. Cell Biol. 37, 825-831.ANDRE, J. (1961). Sur quelques details nouvellement connus de l'ultrastructure des orgamtes

vibratiles. Jf. Ultrastruct. Res. 5, 85-108.BAWA, S. R. (1964). Electron microscope study of spermiogenesis in a fire-brat insect, Thermobia

domestica Pack. I. Mature spermatozoon. J. Cell Biol. 23, 431-446.BEHNKE, O. & FORER, A. (1967). Evidence for four classes of microtubules in individual cells.

J. Cell Set. 2, 169-192.BIARATI, A., JR. (1967). Struttura ed ultrastruttura dell'apparato genitale maschile di Droso-

phila melanogaster Meig. I. II testicolo. Z. Zellforsch. mikrosk. Anat. 76, 56—99.BROSSEAU, G. E. (i960). Genetic analysis of male fertility factors on the Y chromosome of

Drosophila melanogaster. Genetics 45, 257-274.CAMERON, M. L. (1965). Some details of ultrastructure in the development of flagellar fibres of

the Tenebrio sperm. Can. J. Zool. 43, 1005-1010.DAEMS, E. Th., PERSIJN, J. P. & TATES, A. D. (1963). Fine-structural localization of ATPase

activity in mature sperm of Drosophila melanogaster. Expl Cell Res. 32, 163-167.GIBBONS, I. R. (1963). Studies on the protein components of cilia from Tetrahymena pyriformis.

Proc. natn. Acad. Sci. U.S.A. 50, 1002-1010.HESS, O. & MEYER, G. F. (1968). Genetic activities of the Y chromosome in Drosophila during

spermatogenesis. In Advances in Genetics, vol. 14 (ed. E. W. Caspari), pp. 171-223. New Yorkand London: Academic Press.

KAYE, J. S. (1964). The fine structure of flagella in spermatids of the house cricket. Jf. Cell Biol.25, (no- 3- part 2), 31-39-

KIKFER, B. I. (1966). Ultrastructural abnormalities in developing sperm of X/O Drosophilamelanogaster. Genetics 54, 1441-1452.

KIEFER, B. I. (1968). Y-mutants and spermiogenesis in Drosophila melanogaster. Genetics 60, 192.KIEFER, B. I. (1969). Phenotypic effects of Y chromosome mutations in Drosophila melanogaster.

I. Spermiogenesis and sterility in KL-i~ males. Genetics 61, 157-166.MEYER, G. F. (1968). Spermiogenese in normalen und Y-defizienten Mannchen von Drosophila

melanogaster und D. hydei. Z. Ztllforsch. mikrosk. Anat. 84, 141-175.PHILLIPS, D. M. (1966). Substructure of flagellar tubules. J. Cell Biol. 31, 635-638.RENAUD, F. L., ROWE, A. T. & GIBBONS, I. R. (1968). Some properties of the protein forming

the outer fibres of cilia. J. Cell Biol. 36, 79-90.SATIR, P. (1967). Morphological aspects of ciliary motility. In The Contractile Process, pp. 241-

258. Boston: Little, Brown and Co.SHELANSKI, M. L. & TAYLOR, E. (1968). Properties of the protein subunit of central-pair and

outer-doublet microtubles of sea urchin flagella. J. Cell Biol. 38, 304-315.

(Received 11 April 1969)

Drosophila sperm flagellum

ABBREVIATIONS ON FIGURES

a

afapatb

cctder

A fibre (microtubule) of peripheraldoubletaxial fibres (microtubules)projection from accessory microtubulesaccessory microtubuleB fibre (microtubule) of peripheraldoubletcentriolecentral microtubules of '9 + 2'dense non-tubular portion of satelliteendoplasmic reticulum

/

mx

mt

nP

Ptssa

sf

core ' fibre' of central and accessorytubuleslarger Nebenkern derivativesmaller Nebenkern derivativenucleusprojection from B fibre which developsinto accessory tubuleperipheral microtubules of ' 9 + 2'spokessatellitessecondary fibres

186 B.I. Kiefer

Fig. i. Cross-section of main piece of mature sperm. The dense cap-like structure is themitochondrial element (mj derived from the Nebenkern. A second mitochondrial ele-ment (nij) degenerates in this species. The axial fibre complex is composed of central(ct) and peripheral (pt) microtubules, satellites (sa), spokes (s), and secondary fibres(sf) Arrows indicate connexions between A-fibre arms and adjacent B fibres and/orsatellites, x 140000.Fig. 2. Axial fibres (af) developing from complex centriole (c). Lines indicate approxi-mate region of the cross-section shown in the inset. The centriole has the typical tripletconfiguration of the peripheral tubules, x 54000; inset x 100000.

Drosophila sperm flagellum 187

188 B. I. Kiefer

Fig. 3. Initial formation of the axial fibre complex in an early spermatid. Only peripheralmicrotubules are present and there is a projection (p) extending from each of the Bfibres, x 145000.Fig. 4. Slightly later stage of maturity than that of Fig. 3. Central microtubules are pre-sent and spokes (s) are beginning to form, x 145000.Fig. 5. The accessory microtubule (at) is formed from the B-fibre projection (arrows)by a process roughly equivalent to rolling a sheet into a tube. This section is from amutant that shows defective mitochondrial development, x 120000.Fig. 6. Slightly later stage of maturity than that seen in Fig. 5. The accessory tubules arecomplete and are beginning to give rise to a projection (ap). Dense material (d) is form-ing next to each accessory tubule. The spokes and secondary fibres are clearly definedand the inner arm of the A fibres connects adjacent doublets. At some points (arrows)there appears to be a branch of this arm directed inward, x 120000.

Drosophila sperm flagellum 189

er

er

•c*

0-1-7/m

190 B. I. Kiefer

Fig. 7. Considerably later stage of maturation. The accessory and central tubules arenow dense and contain a ' fibre' in their cores (/). Adjacent satellites (accessory tubuleplus dense non-tubular material) are connected by the accessory tubule projection (ap).x 120000.

Fig. 8. First stage of degeneration. Separation of adjacent satellites (arrow heads) andloss of connexion between secondary and central fibres (arrow), x 120000.Fig. 9. Complete loss of satellite connexions and total disruption of the axial fibrecomplex. Note that satellites, doublets, spokes, and secondary fibres remain together asunits, x 54000.

Drosophila sperm flagellum

192 B. I. Kiefer

Fig. 1 o. The next stage in the degenerative process is the loss of the inner portion of theB fibre (arrows), x 120000.

Figs, i i , 12. Subsequent loss of A fibres, the remaining portion of the B fibres, thespokes, the secondary fibres, and the non-tubular portion of the satellite. The outerportion of the A fibre is lost first (1) and the remaining portion of the B fibre is stillpresent after the entire A fibre has been lost (2). An outer rim of the non-tubular por-tion of the satellite remains (3). This appears to be continuous with the accessory tubulebut should not be confused with the projection depicted in Figs. 6 and 7 which is onthe other side of this tubule. Fig. 11, x 160000; Fig. 12, x 120000.

Fig. 13. Late stage of degeneration of the axial-fibre complex. All that remain are thecentral (ct) and accessory (at) microtubules with the outer rim of the non-tubularportion of the satellite (arrow), x 240000.

Drosophila sperm flagellum 193

C E L 6

194 B. I. Kiefer

Fig. 14. Material remains attached to the accessory tubule projection (arrows) after theaxial-fibre complex has been disrupted. This material appears as a double structure andis presumed to represent most of the non-tubular portion of the adjacent satellite,x 140000.

Fig. 15. A triple linkage between adjacent peripheral groups is shown between thearrows. The inner A-fibre arm connects to the next B fibre, the outer A-fibre armconnects to the next accessory tubule, and the accessory tubule projection connects tonon-tubular portion of the adjacent satellite Gust beginning to form in this case),x 200000.

Fig. 16. Two pairs of central fibres which have come to lie next to each other after dis-ruption of the axial-fibre complexes. The fact that they have moved as pairs suggeststhat they are physically linked, x 140000.

at

Fig. 17. A composite diagram of a portion of the axial-fibre complex, showing thestructural relationships among two peripheral units and the central pair of micro tubules.