The first description of the 9+2 subfibrillar structure of flagella/cilia came from a short series of experiments on the morphology of spermatozoa from three species of animals in Melbourne during the third quarter of 1948. Earlier in that year 1 had commenced a Masters Thesis in the Department of Zoology of the University of Melbourne. Although T had won a Prize Research Fellowship to support me in my M.Sc. studies, the stipend was modest and I was poor. The CSIR (later to become CSIRO) Poultry Research Centre at Werribee (near Melbourne), with whom previously I had spent three months as a temporary employee during the University vacation, offered a half-time salary to supplement my Prize stipend if I chose to work on sperm physiology as my research project. I discussed this proposition with my supervisor, Professor Oscar Tiegs, FRS, who had no objection. He pointed out that if I tackled this topic 1could expect no expert help from other members of the staff of the department, since none was specially knowledgable about sex physiology. With all the arrogance and ignorance of my twenty-two years however, I did not think that posed a problem. So, with some largesse from CSIRO in my pocket and several Rhode Island cockerels, supplied by CSIRO, in the backyard of the Zoology Department, I set out to study the physiology of fowl sperm. It was my pious hope that a better understanding of sperm biology might lead to an improvement in fertility of breeding fowls. Within the first few months I had made some novel observations on the influence of tonicity, p H and oxygen tension on sperm motility in v i m and decided that it might be useful to see if I could correlate motility changes with alteration of sperm structure. The Department of Zoology had a strong morphological tradition, due mostly to Tiegs and his immediate predecessor, the cytologist Professor W. E. Agar, FRS, and as students we were encouraged to think of structure when considering function. T had looked at live fowl sperm with the new phasecontrast optics and also at fixed and stained specimens in various ways, but there was not too much to be seen of the detail of these cells. The diameter of the flagella was close to the limit of resolution of visible light. I had heard of a new electron microscope at the CSIR Division of Industrial Chemistry across town and

through Professor Tiegs, I contacted Dr. Lloyd Rees, who led the Section of Chemical Physics of that Division which housed the electron microscope. In turn I was introduced to John Farrant, whose field was electron optics and Alan Hodge, a young physical chemist, a couple of years my senior. The section had one of the early model RCA EMU instruments which, when purchased new a year or two before, had a rcsolution of 100 A.Farrant had already redesigned the objective lenses and in 1948, its resolution was better than 50A. Some 10 years later, Farrant had improved that to better than 2 A . It was agreed that the electron microscope could be of assistance to my project - so a little later I collected some fowl sperm and took them down to Fishermans Bend where the CSIRO laboratories lay. In the late 1940's, the electron microscope was of limited usefulness in biology. Objects such as viruses, bacterial fimbrae, fragments of whole cells could be described, but little structural detail was visible in whole cells, even those as small as bacteria. Nevertheless, at this time the CSIRO electron microscope group in Melbourne made some interesting and basic contributions to the structure of muscle by studying fragments of myofibrils. It was obvious that ultra-thin tissue sections were required, if the electron microscope was to be of major usefulness in biology and many labs were trying to devise methods to generate them. Some, such as Pease and Baker(') attempted to adapt and improve standard microscopic sectioning methods. I had an old Spenser microtome, adapted according to Pease and Baker's description. and cut sections of various tissues - including muscle, and examined them in the EM - but they were still too thick to show any fine detail. A breakthrough occurred around 1952, when new imbedding materials were introduced mainly in the USA, together with glass knives and various effective ul tramicrotomes. From that point on biological electron microscopy really took off as an important part of cell biology. Improved fixation method5 followed a little later. Ballowitz(2),in his pioneering studies on the structure of bird sperm published 60 years before, had macerated his sperm in glycerine to reveal the presence of subfibrils in the sperm tail. I thought this too crude a procedure for electron microscopy and it was not used in our first studies of the structure of flagella. (A year or two later, when my experience as a researcher had matured a little, T was more pragmatic and found glycerine a useful medium for examining other structures in the sperm midpiece and head with the ordinary microscope.) At the Fishermans Bend laboratories, I learned by direct experience that whole cells (even small sperm cells) were opaque to the electron beam. Since effective ultramicrotomy was still some years in the future, other methods of microdissecting our sperm had to be devised before we could unlock their inner structural secrets. I suggested that we use osmotic shock to open up the cells

and digestion with cpccific enzymes to aid the fine dissection of structure and its chemical characterisation. About this time, Dawson and MacFarlane(') had demonstrated the effectiveness of proteases in changing the electron density of components of Vaccinia virus. Both procedures worked well the first time we tried them with fowl sperm and almost from the first day many fascinating details of structure could be seen clearly enough to be described. Within 6 weeks of starting the project, we had collected sufficient data to sit down and write a first draft of our paper on sperm morphology. It was the first research paper I had written. I remember thinking, doing research is fun and it is dead easy. Research to me always has been fun serious fun - but rarely ever again was it easy. Sperm consist of three main components - a head surmounted by an acrosome, a midpiece and a long tail which is a flagellum (Fig. 1). We found that in the fowl sperm, the dagger shaped acrosome was encased in a membranous acrosomal cap which was dislodged by

Ahhreviationy used in figures. a , acrosome: ac, acrosomal caps; as. acrosomal spine; ff, fine filamenls resistant Lo trypsin: h, head of sperm; L , L fibrils: rn, sperm midpiece; M , M fibrils; n r , narrower region of fibril: SJ L fibrils terminally truncated by pepsin; s L , sheet of L fibrils; t. tail flagellum; ir, tip of tail flagellum. Fig. I. The fowl spermatozoan. Electron microscopic pictures of fowl spermatozoa taken at low magnification to show the general structure.

osmotic shock (Fig. 3B,C). This acrosomal cap was resistant to digestion by trypsin, but not to pepsin. The long, narrow, somewhat cylindrical head, which contains the nuclear DNA, was quite opaque to the electron beam but was opened up by tryptic digestion. The sperm midpiece consisted of a mitochondrialderived structure wound around the anterior extension of the bundle of filamcnts constituting thc tail flagellum. The sperm tail turned out to be of most interest. The tail flagellum consisted of 9 subfibrils associated laterally to form a hollow cylinder around a further 2 subfibrils, which were different in structure and chemical composition from the other 9. This fibrillar complex was invested by a membranous sheath having no particular structural features (Fig. 2D). In this characteristic, the sheath differed markedly from that of bull and human sperm. The flagellum sheath of these two species of mammalian sperm contained a tightly wound helical fibril resistant to osmotic shock and to digestion with pepsin(')). Osmotic shock opened up the tail sheath of the fowl sperm allowing the 11 fibrils there contained to spread out on the support film (Fig. 2A). We called the 9 uniform fibrils the L fibrils and the 2 which occupied the inner lumen the M fibrils. The former seemed to be differentiated into two components one of which was more elastic and did not fracture upon drying. Occasionally the appearance of these L fibrils was of a series of short narrower regions distributed randomly along the fibril (nr in Fig. 2A). Upon digestion with trypsin, the L fibrils appeared to bc reduced in diameter to that previously observed in the narrower regions (See Fig. 2D, 3A). The M fibrils had no sign of this more elastic component and were completely digestcd by either trypsin or pepsin. Although we had no practical recourse to examining structure by sectioning the sperm - we tried, but our sections were too thick to display any useful detail - we were confident that the 9 L fibrils formed the walls of a hollow cylinder. We had several reasons for believing this. The most direct evidence came from observing flagella partially split open lengthwise showing the fibrils and their longitudinal associations. Our photographs of partially split flagella however, (Fig. 2C-1), did not have a resolution as good as the technical excellence of some of the others and for this reason my collaborator Alan Hodge refused to use them in our paper. We did have many pictures of flagella completely split open, end for end, revealing sheets of 9 or less, L fibrils, one of which was used in the paper (Fig. 2C-2), - since it was of approved technical quality! We used only two enzymes in our study - pepsin and trypsin - and they produced quite different effects. Pepsin, although an endopeptidase. attacked the L fibrils from their posterior extremities in a processive fashion so that after a certain period of digestion all the L fibrils were truncated, but to the same extent (Fig. 2B). , Trvmin, on the other hand. seemed to digest \

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away some component/s of the T~, fibrils along their whole length, leaving very thin sub-fibrils one fifth to one tenth the diameter of the L fibrils (Fig. 2D and 3A). In considering the possible functions of the two types of subfibrils it seemed likely that the L fibrils were the contractile elements. In our 1949 paper, we suggested that the 2 inner M fibrils had a conducting function, transmitting down the flagellum a coiitractile stimulus to allow a synchronous and progressive contraction along the 9 L fibrils. The later observation by Randall’s laboratory of paralysed Chlamydonionas mutants having an abnormality of the centre 2 filaments gave some credibility to this idea. Trypsin, unlike pepsin, also attacked the cell membrane of the sperm head and weakened it so that its contcnts wcre releascd upon drying on the support film of the electron microscope sample grid. thus revealing details of structure of the spine-like acrosome (Fig. 3A). As I mentioned before, trypsin affected neither the conical acrosomal sheath nor the flagellar sheath. Alan Hodge and I were both concerned about the possibility of artifacts distorting or invalidating our measurement of particular structures, or indeed of their reality. One concern was the distortion of the dimensions of a cell or cell organelle when it dried down on the support membrane - the dimensions measured would tend to bc differcnt from what they were in living cells. We carried out a series of careful measurements on whole sperm and osmotically shocked sperm, spread on support membranes on grids, then frozen in liquid nitrogen before dehydration to prevent any flattening by surface tension forces or shrinkage by dessication at room temperature. In general, the results of this study did not change the general conclusion we had reached before, and although there was a little flattening of fibrils etc, it was sufficiently minor to be ignored as a major problem and we did not elaborate on it in our paper. Another type of artifact which to me was more likcly, was a changc in structure of the mitochondrial elements in the midpiece during preparation for electron microscopy. In the days before phase optics were readily available and differentiation of cellular detail relied mostly on the efficacy of cell fixation and microscopic stains, mitochondria and mitochondrialrelated devised structures wcre recognised as somewhat labile entities. For that reason, at the earliest opportunity, but after our electron microscope (EM) paper was published, T attemptcd to confirm our EM results with observations on ’live’ sperm using phase optics and a suspending fluid having a higher refractive index than water. Our EM study had concluded that the midpiece contained a number of flattened spherical mitochondrial elements distributed around an axial filament (the anterior continuation of thc tail flagellum). Earlier studies by light microscopes using certain staining methods produced an image of a helical mitochondrial structure in the midpiece of bird sperm.

A year after we published our EM paper, 1 obtained somc excellcnt pictures of a helical structure in the midpiece of unstained ‘live‘ sperm suspended in a glycerol-saline solution. Glycerol was included to increase the refractive index and allow visual ‘penetration’ of the midpiece membrane to show internal structure. Therefore, it seems probable that the structure of thc midpiece as determined by our E M study was wrong and the spherical structures described were the result of a preparation artifact. Alan Hodge and 1 wrote up a draft of the paper - my own first(’). We split the chore - I wrote the Tntroduction, he wrote the Methods section and a first draft of the Results. We wrote the Discussion jointly. Previously, I had turned up a number of interesting classic papcrs on the structurc of bird spermatozoa mostly from the school of the great German morphologist Ballowitz(2) and the Swedish morphologist Retzius(‘). The latter, a man of independent financial means, published his researches privately in great folio volumes which fortunately were available at the Melbourne Public Library. The first draft of the paper arrived painlessly and both of us were rathcr pleased with it. The pictures were pretty although I thought we had too many of pepsin-digested sperm. and I regretted that some biologically informative but technically inferior pictures such as Fig. 2C-1 were left out. At the very end, when 1 thought we had agreed on the final draft, Alan. without mentioning it to me. rewrote a paragraph of ‘my‘ Introduction in which I had recalled Hallowitz’s obscrvation that the flagella of sperm tails of various finches, when macerated and stained with iron haematoxylin, could be seen to consist of a number of subfilaments. Sometimes there were 2 or 7, and sometimes 10 or 11 subfibrils. In his rewrite. Alan complained that since these subfibrils in the tail flagcllum of our bird sperm were only about 400A in diameter they were well below the limit of resolution of the light microscope. Obviously then it was likely that Rallowitz’s description owed more to imagination than to fact. This embroidery to the fabric of our paper was not noticed by me and without checking the M/S further I passed it on with some pride to the head of my Department and my wpervisor, Professor O.W. Tiegs, for rcview. Unfortunately for us, Tiegs was a fan of BallowitL, one of the 19th century giants of descriptive biology and morphology. and when 1 presented him with my first creation, his reaction after reading the Introduction was rather violent. Normally a quiet and gentle person, he was roused to a passion by our transgressioii of good bchaviour - words shot out such as, ‘young pups’, ‘new technique‘. ‘libel one of the greatest biologists who ever lived’ - ‘young men have been disinissed from their departments for smaller crimes‘. He simmered down when I protested my ignorance of the prcsence oi the offending sentencc. When it was substituted by a more graceful tribute to Ballowitz’ genius as a morphologist he relaxed sufficiently later to congratulate me on our

Fig. 2. Structure of the sperm flagellum. A. Details of the internal structure of the sperm flagellum showing 9 fibrils ( L )of similar dimensions and appearance plus 2 other smallcr fibrils ( M ) differing in structure and appearance. The sperm were osmotically shocked hcfore being spread on a support film, dried and shadowed with platinum. Note the occasional narrower regions suggesting a substructure in the L fibrils. B. Effect of pcpsin on flagella. Note the absence of the two smaller A4 central fibrils and the terminal truncation of the I , fibrils. The sperm were fixed in formalin before being digested in pepsin, spread on a support film, dried and shadowed with platinum.

effort! (In his assessment of Ballowitz‘s description of finch sperm tails, Alan did not recognize the nature of the ‘lake’ staining process used by the former - which had the effect of greatly increasing the diameters of these fine fibrils).

The paper was accepted without alteration and published in a new Australian journal - the Australian Journal of Scientific Research Series B in 1949(5). Perhaps it would have attracted a wider audience had it been published in one of the established international

Fig. 2. C. Sperm flagellum partially ( c i ) , or fully ( ~ 2 ) splil . open by osmotic shock showing the tube like disposition of the L fibrils. Staincd with phospliomolybdic acid. D. Trypsin treated sperm showing the intact membranous tail sheath (ts) and the sperm midpiece (m) separated from the head. Many trypsin resistant filaments ([fl thought to be derived from the larger L fibrils are present.

journals. It was noticed by some, including Prof. Irene Manton the plant cytogeneticist, who later described a 9+2 subfibrilla structure of flagella/cilia in fern sperm in the first of a series of papers on the subject published in 1951(7).Unfortunately she did not acknowledge our effort. She did so afterwards, however. in private correspondence to me - and in publications soine years on. A few months later in 1949, Alan Hodge, using the

methods we had pioneered for fowl sperm cells. described a similar 9+2 structure of the tail flagella of bull and human sperm('). Little did we know at that time that we had discovered a biological constant. Subsequently, and following the development of efficient methods of cutting ultrathin sections for electron microscopy, the 9+2 structure turned out to be rather universal for

Fig. 3. Structure of the fowl sperm acrosome. A. Effect oCdigestion with trypsin on fibrils of thc tail flagellum, head and acrosome of sperm. Note the fine filaments which are about one tenth of the diamctcr of the larger L fibrils and which we thought were a component of the latter. The head membrane and its contents were darnaged to varying degrees, but thc acrosome was intact. Stained with phosphomolybdic acid. B. Acrosomal region of the sperm head. Osmotic shock has dislodged the acrosomal cap or membrane from the anterior spine of the acrosome - the cap i s shown partly rcinoved. C. Detail of the acrosomal spine and anterior portion of the head. The acrosomal cap was displaced by osmotic shock. Staincd with phosphomolyhdic acid. These figures are from E.M. photographs taken during thc initial study in 1948. All cxcept Fig. 2c and 2d were published previously in reference 4.

cilia/flagella from both the animal and plant kingdoms@). The couple of years I spent studying fowl sperm did lead, a little later, to some improvements in artificial

insemination and fertility in fowls, but by this time 1had a scholarship to Cambridge and had commenced a new research career in the field that became known as molecular genetics.

Acknowledgements Dr. Robin Holliday FRS persuaded me to write this reminiscence and then commented on it for me, for which I am grateful. I thank the Manager, CSIRO Editorial Services, for permission to include in this article Figs 1,2a,2b,3a,3b,c, previously published in the Aust J . Sci. Res. B 2, 271-286 (1949).

3 DAWSON. I. M. -\mMACFARI A N F . A. S. (1948). Structure of an animalvirus. &‘a‘alur~161. 464-466. 4 HODGF.,A . J. (1949). Electron niicrowopic studies of spermatozoa. Au.sira/iun .I. Sci. Rci. (B) 2 . 368-178. 5 GRIGC.G. W. A N D HODGF,A . .I. (1949). Electron microscopic attitlie> of spcrniatoroa. Ausiridian .I. 9;. Rr.i. (B) 2. 271-286. 6 RETZITR, G. (1909). Die Sperniieri der Vogel. B i d . LtntfJrmch 11. 89. 7 MAuroN. 1 . A N D CI A RK E. H. (‘lqSl), Lkmonstration ot compound cilia in a fern spermalomid wJith the electron rnicroscopc. .I. ESP. Bor. 2. 125-28. 8 GIBBONS, 1.K. (1981). Cilia and tlaqlla ofenkaryotes. J. Cell B i d . 91. (3 Par1 2). 107s-124s.

References 1 PEASE,D. C. AND BAKER* R. F. (1948). Sectioning techniques for electron microscopy using a conventional microlome. Proceedings of The Society of For Experimental Biology and Mrdicine. 67, 470-474. 2 BA~.I.OWITZ, E. (1888). Untersuchungen uber die Struktur dcr Spermatomen. Arch. mikr. Anar. 32. 401-73.

Molecular Biology, Division of Biomolecular Engineering, PO Box 184.North Ryde, NSW 2113.

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The first description of the 9+2 subfibrillar structure of flagella/cilia came from a short series of experiments on the morphology of spermatozoa fro...
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