Direction of Sliding and Relative Sliding Velocities Within Trypsinized Sperm Axonemes of Gallus Domesticus

Direction of sliding and relative sliding velocities within trypsinized sperm axonemes of Gallus domesticus DAVID M. WOOL LEY and ANDREW BRAMMALL* Department ofl'liysioloi>y. The Medical School, L'niversity of Bristol, Bristol BSS ITD, L'K •Present address: MRC Experimental Embryology and Teratology Unit, Medical Research Council Laboratories, Carshalton, Surrev SMS 4EF, UK Summary Trypsin digestion of demembranated fowl sper- orderly way by inter-doublet sliding. All the matozoa caused a longitudinal splitting of the doublets of the axoneme could be reactivated and distal part of the axoneme. The resulting strands, in all instances the direction of sliding was the consisting of groups of doublet microtubules, same as that reported for the cilia of Tetrahy- formed left-handed helices. On the evidence of tnena. Within the groups of doublets the electron micrographs, the digestion had caused measured inter-doublet displacements were gen- the loss of dynein arms from the outer row; it is erally similar, suggesting that the rates of sliding assumed that the doublets remained linked had been equivalent. These findings are con- together by dynein arms of the inner row. When trasted with the differential pattern of activation such helices were mechanically detached from that is assumed to occur in vivo. the proximal flagellum and reactivated with ad- Key words: Gallus domestic/is, sperm axonemes, sliding enosine triphosphate, they lengthened in an velocity. Introduction We have therefore made use of these preparations to study two aspects of the sliding behaviour relevant to It is well established that the motion of eukaryotic cilia the question of control and coordination. First, we and flagella depends upon active sliding between the have confirmed the important finding of Sale & Satir doublet microtubules of the 9+2 axoneme. The rel- (1977) that the active sliding has a single polarity under evant ATPase activity resides in the dynein proteins these circumstances. Second, we have measured the that project as inner and outer rows of arms from the A within-group displacements to discover whether the individual doublets achieve similar sliding velocities; tubule of each doublet and interact cyclically with the B these data augment the short series published for sea- tubule of the adjacent doublet (reviewed by Gibbons, urchin sperm axonemes by Takahashi el al. (1982). 1981). The control, spatial and temporal, of this sliding Finally, from the absence of the outer row of dynein so that it gives rise to a pattern of bending, bend arms from our electron micrographs, we attribute the propagation and oscillation remains to be elucidated. sliding behaviour of these preparations to the activity of In this work we have used spermatozoa from the the inner row of dynein arms. domestic fowl (Gallus domesticus) as a convenient source of long lengths of simple 9+2 axoneme. Apply- Materials and methods ing the standard procedures to induce active sliding and axonemal disintegration (Summers & Gibbons, Collection of spennatozoa 1971), we noted that exposure to trypsin caused Cocks of a commercial broiler strain (Ross) were obtained at longitudinal splitting of the axoneme and that sub- 50 weeks of age as ex-breeding stock. They were fed a pelleted sequent treatment with ATP produced an orderly diet and maintained on a 16 h: 8 h (light/dark) cycle. After an sliding apart of these subsets of the original nine initial period of training, the most productive animals were doublets. kept and used as semen donors every 2 or 3 days. The Journal of Cell Science 88, 361-371 (1987) Printed in Great Untam © The Company of Biologists Limited 1987 361 collection of semen by massage followed the standard To obtain thin sections for electron microscopy, the cells or 2-operator technique described by Lake & Stewart (1978). fragments were routinely fixed for 30 min in suspension by Immediately after collection into graduated 10-ml glass adding an equal volume of 5 % glutaraldehyde in 01M- tubes, the semen was held at 40°C, then allowed to cool to sodium cacodylate, then centrifuged (2000 £ for 20min), room temperature over 30min. Samples that were noticeably washed in buffer for 20 min, treated with 1 % OsO.) in the contaminated with faecal and urinary excretion were dis- same buffer for 1 h, dehydrated in ethanol and embedded in carded. In general the semen was not pooled before use. The Epon 812 by conventional methods. In addition, some 'ejaculates' thus obtained ranged in volume from 015 to preparations were fixed in a Hepes-buffered glutaraldehyde 1-0 ml and contained typically about 109 spermatozoa (sperm solution containing 1 % tannic acid, exactly according to the density was not measured routinely). method of Warner et al. (1985). Silver-gold sections of the pellets were stained with uranyl acetate and lead citrate and examined in a Philips EM300. Preparation of gmups ofaxonemal doublets To prepare negatively stained axonemes, the fixed suspen- A stock suspension of spermatozoa was initially made by sions were first dialysed against distilled water for 2-3 h to diluting the semen 1:20 (v/v) in Hanks' balanced salt remove the salts. The droplets were placed for 4 min on 300- solution at room temperature. (For observations on the mesh grids (Pioloform-coated, carbon-stabilized), which had motility of intact sperm the Hanks' solution contained been treated immediately before with an aqueous solution of 3mgml~' of bovine serum albumin.) Demembranation was poly-L-lysine (l-5mgml~') rinsed off with de-ionized water. achieved by adding O'OS ml of the sperm suspension to 1 ml of The suspension was then drained off the grid and immedi- a 'reactivation solution' containing 0-04% Triton X-100. The ately replaced by a drop of negative stain. This was then fully reactivation solution was based on that used for mammalian drained off and the gnd allowed to dry. Uranyl acetate (3 % spermatozoa (Yeung & Woolley, 1984) and contained aqueous), the initial choice of negative stain, was later given 230 mM-sucrose, 15 mM-potassium glutamate, 3mM-MgSO.t, up in favour of a 2% aqueous solution of methylamine OSmM-EDTA and 20mM-Tris • HC1 buffer, adjusted to tungstate (Agar Aids Ltd). The quality of the negatively pH 7-9. Naturalistic flagellar beating could be induced in this stained grids was variable and the yield of well-displayed medium by adding 1 mM-ATP, though it lasted only specimens was low. The extended axonemes were photo- 5-10 min. The longitudinal splitting of the distal axoneme graphed on several plates at between X4500 and X 10000, was achieved by adding 20j(gml~' trypsin to the suspension with higher magnification micrographs of the regions where of demembranated sperm for 4min at 23°C and then doublets terminated. Prints (X2-5 enlargement) were as- terminating the digestion with 40j(gml~' trypsin-inhibitor sembled into montages and estimates of the inter-doublet (from soybean; Sigma). displacements were then made by hand, using a map The split, distal portions of the axoneme were then measurer read to l/16 inch. mechanically detached from the proximal axoneme by several passages through a standard Pasteur pipette. Reactivation of these separated groups of doublets was achieved by adding Results ATP (250/JM-l mM, varying according to preparation). Ac- tive sliding was arrested after 5-15 s by mixing the suspen- General observations on flagellar structure and sion with an equal volume of 5 % glutaraldehyde in 0-1 M- sodium cacodylate (pH 7-4). The results of this were moni- motility tored by dark-field light microscopy before proceeding to The structure of the cock spermatozoon has been electron microscopy. described and reviewed by Lake (1981). As a basis for the present study, we illustrate again the main features Microscopy of the flagellum. Its length is 70jUm. There is a short mitochondrial section or midpiece (4^tm), containing Suspensions of living spermatozoa were placed in 100/mi deep chambers, made by supporting covershps on strips of an elongated distal centriole proximally and beyond double-sided tape. For viewing trypsin-treated axonemes, a this a 9 + 2 axoneme surrounded by nine very short \2-fi\ droplet of suspension was covered by an unsupported accessory fibres (Fig. 1). Distal to the midpiece is the 22 mm X 22 mm coverslip giving a depth of approximately principal piece, a 9 + 2 axoneme surrounded, for the 25 /.im. These preparations were viewed using a Leitz Ortho- first approximately 30fxm only, by a tapering protein- lux II microscope fitted with a X40 oil-immersion planapo- aceous sheath; the sheath is symmetrical in cross- chromat (N.A. l'00), an oil-immersion concentric dark-field section and apparently unattached to the axonemal condenser (N.A. 1-2-1-4), and a heat filter. For photomicro- doublet microtubules (Figs 2, 3). We can add the graphy, short exposures were required to arrest movement information that at the tip of the flagellum the A-tubule and to obviate the drifting of immobile specimens: therefore a flash unit discharging 200 Ws of input energy in 500 f/s was loses its dynein arms and the electron density of its used, as described (Woolley & Osborn, 1984). Images were interior. Subsequently, the B-tubule terminates as a recorded on Kodak Tri-X 35 mm film; development was in loose strand, leaving the A-tubule to continue as a 'Acutol' (Paterson), for twice the normal time. Some images singlet to the tip (Figs 4, 5, 24). The reduced doub- of living spermatozoa were recorded on videotape using the lets are apparently attached to the cell membrane two-colour dark-field technique for three-dimensional analy- (Figs 4, 5). These features of the tip are fairly typical sis of flagellar movement (Woolley, 1981).

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