Structural Heterogeneity of the Axonemes of Respiratory Cilia and Sperm Flagella in Normal Men

Transcription

1 Structurl Heterogeneity of the Axonemes of Respirtory Cili nd Sperm Flgell in Norml Men L. J. Wilton, H. Teichthl, P. D. Temple-Smith, nd D. M. de Kretser Deprtment ofantomy, Monsh University, nd Respirtory Function Lbortory, St. Vincent's Hospitl, Melbourne, Victori, Austrli Abstrct The ultrstructure of norml humn cili nd flgell ws exmined nd quntittively ssessed to determine the norml vritions in the structure of the xoneme. Cilited respirtory epithelil cells nd spermtozo from 1 norml, nonsmoking mle volunteers who hd norml semen prmeters were fixed for electron microscopy. Tnnic cid nd MgSO4 were included during fixtion to enhnce, in prticulr, xoneml components. In 75 xoneml cross sections per smple, the number of outer doublet nd centrl singlet microtubules, outer nd inner dynein rms, nd rdil spokes were recorded. Sttisticl nlysis of the results showed mrked reduction, from the expected vlue of nine, in the numbers of inner dynein rms (men±se, cili, 531±.13; sperm, 5.38±.16) nd rdil spokes (cili, 4.95±.22; sperm, 5.8±.19). The idel xoneme with ll its structurl components ws seen in only.13% of cili nd.8% of sperm tils. Significntly more doublet microtubules (P <.5) nd less centrl microtubules (P <.1) nd rdil spokes (P <.1) were seen in cili thn in sperm til xonemes. Between subjects there ws little vrition in the men number of structure seen per xoneme. However, within ech smple, the vrition ws considerbly higher, prticulrly for the inner nd outer dynein rms nd rdil spokes. The doublet microtubules hd significntly greter stndrd devitions in the sperm tils compred with the cili (P <.1), nd furthermore, significntly greter number of sperm tils compred with cili showed the incorrect number of doublet microtubules (P <.2). In one semen smple, with norml semen nlysis, 2% of the sperm tils showed incorrect numbers of doublet microtubules, rnging from to compred with only 1.3% in cili from this subject. This study hs demonstrted tht the idel xoneme is rrely seen even in norml smples, probbly becuse of the technicl difficulties in resolution nd visuliztion, nd stresses the need for thorough documenttion of xoneml ultrstructure. This work provides norml dt bse for comprison with ptients who hve chronic respirtory disese nd suspected infertility. Introduction The complex ultrstructure of the cili nd flgell of mny species hs been well-described in the pst (for reviews, see 1, Address correspondence to Dr. Wilton, Deprtment of Antomy, Monsh University, Clyton, Victori 3168, Austrli. Received for publiction 2 June 1984 nd in revised form 18 October J. Clin. Invest. The Americn Society for Clinicl Investigtion, Inc /85/3/825/7 $ 1. Volume 75, Mrch 1985, ). Mny fetures of the xoneme, including the orgniztion of microtubules, the outer nd inner dynein rms, nd the rdil spokes, re thought to contribute to its motility. Disorders of the ultrstructurl pttern of cili nd flgell disrupt xoneml motility (3, 4). In humns the co-existence of chronic sinobronchil infections nd mle infertility hs in some ptients been shown to be ssocited with immotile or dyskinetic cili or flgell; this is known s the "Immotile" or "Dyskinetic" cili syndrome (5-7). This syndrome is generlly ttributed to either congenitl bsence of dynein rms, disruption of rdil spokes, or trnsloction of the microtubule doublets (7-9). There re mny cse reports of other humn xoneml bnormlities thought to be ssocited with bnorml motility. However, too frequently, studies hve scribed less clerly defined defects, such s prtil dynein rms (1-14), s the cuse of clinicl condition without supporting dt on its frequency in the smple nd without knowledge of vritions in xoneml structure nd their frequency in norml tissue smples. In recent study, Rossmn et l. (15) compred microtubule bnormlities in xonemes of cili from 3 norml subjects nd 21 ptients with vrious respirtory diseses. In the norml subjects, 2.6% of cili hd peripherl microtubule bnormlities nd.6% hd centrl microtubule defects. However, the frequencies of other xoneml structures, such s dynein rms nd rdil spokes, were not recorded nd the dt were not compred with sperm flgell. To dte there re no detiled comprisons of cilil nd flgellr ultrstructure from norml men, mking it difficult to drw conclusions bout the xoneml ultrstructure of specimens from ptients. This pper presents controlled, quntittive ssessment nd comprison of respirtory cili nd sperm flgell from 1 norml mles using new technique of fixtion to enhnce the ultrstructurl detil of the xoneme. We exmine the vrition in ultrstructure, the types of bnormlities tht occur, nd their frequency in lrge number of xonemes. Methods Subjects. Ten norml, nonsmoking mles were selected for study using the following criteri: no personl or fmily history of chronic respirtory illness, no history of upper or lower respirtory trct infection in the 3 mo prior to study, nd no personl or fmily history of genitl disese or infertility. They lso hd norml mucociliry clernce time nd semen nlysis (see below). The study ws pproved by the Monsh University Stnding Committee on Experiments Involving Humn Subjects nd followed the ethicl guidelines of the Ntionl Institutes of Helth nd Ntionl Helth nd Medicl Reserch Council of Austrli. Subjects prticipted only fter signing form of consent. Collection of tissue nd ssessment of xoneml function. Cilited nsl epithelium ws collected from the inferior turbinte of ech subject using stndrd nylon cytology brush (Olympus BC-9C, Olympus Corp., New Hyde Prk, NY). This method of smple collection does not require locl nesthesi nd does not cuse Structurl Heterogeneity in Humn Axonemes 825

2 Tble I. Averge Age, Semen Prmeters, nd Mucociliry Clernce for IO Norml Subjects Prmeter Men±SD Rnge Age (yr) 23.± Sperm/milliliter ejculte (XJO5) 66.3± Motile sperm/ejculte (%) 68.± Motility index 186 ± Mucociliry clernce (min) 12.3± bleeding (16). Cellulr mteril from the brush ws suspended in smll volume of Dulbecco's Modified Egles medium contining 1 mm NHCO3, 35 mm NCl, nd 1 mm Hepes (ph 7.4, 297 mosm) t 37 C. The specimen ws nlyzed t X 4 mgnifiction nd 37 C using Nikon Diphot inverted microscope (Nikon Inc., Grden City, NY) with ttched tissue culture incubtor. Ciliry motility ws ssessed visully nd ll specimens hd ctively beting cili. In vivo nsl mucociliry clernce ws ssessed by the scchrine trnsit time (17, 18). All subjects hd norml trnsit times of <2 min (Tble I). Semen smples were collected by msturbtion nd the percentge of motile sperm nd the motility index (19) determined within 2 h. Semen smples were regrded s norml nd cceptble for further ultrstructurl nlysis if >5% of the sperm were motile, the motility index ws t lest twice the percentge motility, nd the sperm concentrtion > 2 million/ml (Tble I). Fixtion of tissue. Semen smples nd cilited respirtory cells were fixed in solution of 2.5% glutrldehyde, 2.% prformldehyde,.2% picric cid, nd 2 mm MgSO4 in.1 M ccodylte buffer. Smples were postfixed in 2% OSO4 nd then dehydrted through grded series of lcohols, with the finl lcohol contining tnnic cid (see below). The cells were then wshed in epoxy-propne nd left overnight in 1:1 mixture of epoxy-propne nd epon-rldite. After infiltrtion fdr 4-6 h in pure epon-rldite, the tissues were embedded nd llowed to polymerize t 6'C. Improved xoneml stining using tnnic cid nd mgnesium sulphte. The resolution nd ssessment of xoneml ultrstructure is highly dependent upon the stining nd contrst chieved during fixtion nd processing of the tissue. Mny previous reports describing xoneml bnormlities include electron microgrphs of poorly fixed tissue, mking the structurl components difficult to nlyse nd the dignosis of the tissue questionble. Tnnic cid hs been used to resolve individul microtubulr protofilments (2), wheres MgSO4 hs been shown to improve the visibility of dynein rms (21, 22). By ltering the concentrtion of MgSO4 nd tnnic cid it hs been possible to find combintion of these two compounds which drmticlly enhnces the fixtion, stining, nd resolution of xoneml ultrstructure, in prticulr the dynein rms nd rdil spokes (Fig. 1). The optimum concentrtions re 2 mm MgSO4 in the primry fixtive nd.1% (for flgell) or 1.% (for cili) tnnic cid in the finl lcohol dehydrtion step. Higher concentrtions of tnnic cid were found to be unsuitble becuse the incresed electron density produced poorer definition between microtubule structures nd dynein rms. Tissue sectioning. Thin sections showing gold-to-silver interference ptterns were cut with Reichert OM U3 ultrrliicrotome (C. Reichert A. C., Vienn, Austri), mounted on 15-mesh copper grids, nd stined in urnyl cette nd led citrte. Sections were exmined using Jeol loos electron microscope (Jeol Ltd., Tokyo, Jpn) t 6 kv. Axoneml ultrstructurl nlysis. The number of outer doublet microtubules, centrl singlet fnicrotubules, outer dynein rms, inner dynein rms, nd rdil spokes were counted in 75 xoneml cross sections in mtched smples of norml humn sperm tils nd cili. This number ws determined by the summtion verge method (23), in which progressive men is clculted until further observtions do not significntly lter the men. In the cse of the rdil spokes, which hd the highest vrition (see Results), exmining 75 xonemes ws sufficient to stbilize the men, mking further observtions b.1 Pm Figure 1. Enhncement of xoneml ultrstructure with tnnic cid nd MgSO4. () A typicl cilium seen in routinely fixed tissue. Some dytein rms (rrows) nd rdil spokes (rrowheds) cn be seen but most re difficult to distinguish. (b) A representtive outer dynein rm (lrge rrow), inner dynein rm (smll rrow), nd rdil spoke.1 pm (rrowhed) re shown in this cilium fixed with tnnic cid nd MgS4. This technique enhnces resolution of xoneml components nd gretly increses the number of xonemes suitble for comprehensive ultrstructurl ssessment. 826 Wilton, Teichthl, Temple-Smith, nd de Kretser

3 unnecessry. Compound cili or flgell, i.e., more thn one xoneme within membrne, were lso documented. The tissue ws methodiclly scnned nd ll xonemes tht were in perfect trnsverse section were ssessed. To find 75 flgell it ws necessry to scn t lest two grids of sections. The 75 cili cme from not less thn 15 respirtory cells from t lest two grids. The totl number of structures ws verged to give men frequency of ech individul structurl component per xoneme. All ultrstructurl ssessments were mde by one observer. To minimize ny possibility of observer bis in these ssessments, single blind procedure ws used nd cili smples from ptients with respirtory disese nd low motility semen smples were interspersed with the volunteer cili nd sperm smples. The scoring technique hs been shown to be highly reproducible with n intr-observer vrition of.43,.54, nd 1.44% for the outer dynein rms, inner dynein rms, nd rdil spokes, respectively, nd n inter-observer vrition of.43, 2.7, nd 1.9% for the sme three components. To be counted the dynein rms hd to show definite hook projection from the doublet microtubule. Nexin linkges, which join djcent microtubule doublets, re difficult to visulize in humn xonemes nd were not ssessed in this study. To void confusion between the nexin linkges nd inner dynein rms, structures tht rn in direct line between djcent microtubule doublets were not counted. Rdil spokes hd to be continuous from the doublet microtubules to the sheth surrounding the centrl pir. Exmples of how the xoneml structures were identified nd defined re presented in Fig. 2. Clerly both of these xonemes hve nine doublet microtubules nd two centrl microtubules. The cilium in Fig. 2 hs nine outer dynein rms but the inner dynein rms nd rdil spokes ssocited with doublets 6 nd 7 re not clerly visible. The other inner dynein rms nd rdil spokes re visible nd would be counted. The sperm til in Fig. 2 b hs outer dynein rms on ll except doublet 4, nd is missing the inner dynein rm on doublet 5 nd the rdil spoke ssocited with doublet 6. In xonemes where n incorrect number of doublet microtubules existed, e.g., 1 + 2, 6 + 2, etc., the number of outer nd inner dynein rms nd rdil spokes observed were djusted to frction of 9. This prevented the bnormlity of microtubules crrying over to bis the number of dynein rms nd rdil spokes. For exmple, consider n xoneme with six doublet microtubules (insted of the expected nine) in which only four outer dynein rms re visible. The number of outer dynein rms would be converted to 4/6 X 9 = 6 outer dynein rms, i.e., the number of dynein rms tht would be expected if nine doublets were present. This conversion procedure would lso be used to correct for numbers of inner dynein rms nd rdil spokes in such n xoneme. At the distl tip of n xoneme the microtubulr pttern becomes 9 +. In order not to be documented s bnorml, trnsverse sections from this region of the xoneme were not ssessed. Sttisticl nlysis. Becuse both cili nd sperm tils were observed from ech subject, the dt were pired before sttisticl comprisons. Ech SD resulted from 75 observtions, nd so, pired Student's t test ws quick nd pproximte method of clculting significnce levels. A more precise test, the -2 In X method, confirmed ll sttisticl comprisons. Becuse the SEs were very low nd the vrition within ech smple ws considerbly higher (see Results), the dt were lso nlysed fter being pooled. Results The ultrstructurl nlysis showed tht there ws considerble devition from the expected theoreticl vlues of nine outer dynein rms, inner dynein rms, nd rdil spokes (Fig. 3). Significntly more microtubule doublets (P <.5) nd significntly less centrl pir microtubules (P <.1) nd rdil spokes (P <.1) were seen in cili thn in sperm flgell. No significnt difference ws found between cili nd sperm flgell for the outer or inner dynein rms. The results were similr irrespective of whether the dt were pooled or nlysed s mens from individul smples. Although the men vlues for ech smple were very similr, nd consequently, the SE vlues were low (Fig. 3), the b.1 Pm Figure 2. Definition of xoneml components. () A cilium with rbitrrily numbered doublet microtubules, ech showing hooked outer dynein rm. All other structures re clerly defined with the exception of the inner dynein rms nd rdil spokes ssocited with.1 pm doublets 6 nd 7. (b) A sperm flgellum with no visible outer dynein rm on doublet 4 or complete rdil spoke ssocited with doublet 6. No hooked inner dynein rm cn be identified on doublet 5. All other structures would be counted. Structurl Heterogeneity in Humn Axonemes 827

4 z X Z 4 W 3 UI. Fr S CILIA MSPERMTAILS OUTER CENTRAL OUTER INNER RADIAL DOUBETS PAIRS DYNEIN DYNEIN SPOKES Figure 3. Men±SE number of structures per xoneme (unpooled dt). The number of inner dynein rms nd rdil spokes seen per xoneme ws considerbly less thn the expected vlue of nine. Significntly more outer doublets (P <.5) nd less centrl pirs (P <.1) nd rdil spokes (P <.1) were counted in the cili compred with the sperm tils. *, P <.5; **, P <.1. vrition within ech smple ws considerbly higher. This is reflected in the SDs of ech component ssessed for individul subjects (Fig. 4). The SDs of the microtubule structures, i.e., the doublets nd centrl pirs, were low when compred with the outer nd inner dynein rms nd rdil spokes. The SD of the men number of microtubule doublets ws significntly greter in the sperm tils thn in the cili (P <.1). The SDs of the other structures did not differ in the cili nd sperm tils. The idel xoneme in which ll dynein rms nd rdil spokes were visible ws seen in only.13% of cili nd.8% of sperm tils. The microgrphs presented in Fig. 5 demonstrte some of the xonemes seen throughout the smples. Abnormlities of the orgniztion of microtubules were rre in cili existing in only 4.1% of xonemes exmined. They were more common in sperm t 9.9% (P <.2). This dt includes one prticulr semen smple in which 2% of the flgell demonstrted n inpproprite number of microtubules rnging from to When the results were verged for the 75 flgell observed in the smple bove, the mens were not significntly different from norml t 8.89 for the doublet microtubules nd 2.5 for the centrl singlet microtubules. This semen smple hd norml functionl prmeters of 62% motile sperm with motility index of 157, nd in the corresponding smple of cili, only 1.3% of xonemes showed bnorml number of microtubule doublets. Even when this smple ws excluded, there were still significntly more xonemes with microtubule bnormlities in the sperm compred with the cili (P <.2). Abnorml numbers of centrl pir microtubules were found in 1.3% of cili nd 2.4% of flgell (P <.5). Compound xonemes were rre, occurring in only. 13% of cili nd.4% of sperm tils. Discussion This study provides detiled nlysis of xoneml ultrstructure in sperm nd cili from norml, nonsmoking men. It hs shown the devition from theoreticl vlues nd comprison of cili nd flgell from the sme subjects. The demonstrtion of reduction in the number of outer nd inner dynein rms nd rdil spokes in nondisesed tissue is importnt nd my be ttributed to two fctors: () the z 4c 44 z4c I- (A * * -8 *s I2: O OUTER DOUBLETS CILIA.. OUTER * DYNEIN +~S- CENTRAL PAIRS I T ** -- INNER DYNEIN -- S * RADIAL SPOKES Figure 4. SDs of structures in individul smples, so tht ech of the 1 dots in column represents the SD of tht structure in one subject. **P <.1. The vrition between subjects of the men frequencies of structures per xoneme ws low (from Fig. 3), but 2.6r * *6-4 2 C)L OUTER DOUBLETS SPERM TAILS ** I + I * CENTRAL PAIRS. OUTER DYNEIN INNER DYNEIN RADIAL SPOKES within subjects the SDs were lrge, prticulrly for the outer nd inner dynein rms nd rdil spokes. There ws significntly greter SD in the outer doublets of the sperm tils compred with the cili. *. 828 Wilton, Teichthl, Temple-Smith, nd de Kretser

5 b c d 1- N. y In%-C.1pm. M P t PuM.1 pm heds) re present. (c) Sperm xoneme, with its surrounding fibrous sheth (fs). (d) Sperm flgellum with two extr pirs of centrl singlet microtubules (rrows). Figure 5. Some of the bnorml xonemes observed in norml tissue. () Cilium with no well-defined dynein rms or rdil spokes. (b) Cilium showing disrupted circulr rrngement of doublet microtubules. Some dynein rms (rrows) nd rdil spokes (rrowtechnicl difficulties in preserving nd visulizing such smll components, nd (b) the full complement of these structures rrely occurs. It is impossible to isolte the contribution of either of these fctors but it is likely tht the technicl problem of fixtion nd resolution of the tissue plys the mjor role. This hs been noted previously in cili from 1 "norml" subjects (22) when only 31.3% of the expected totl number of dynein rms could be observed. This ws considerbly less thn the number we recorded nd could be becuse four of the subjects in the other study hd chronic sinusitis nd one hd llergic rhinitis. The difficulty in visulizing dynein rms must be considered when disesed tissues re described s hving, for exmple, "prtil dynein rms" (1-14). Such rther obscure terminology should be used with cution nd only fter the exmintion of mny xonemes. Becuse of the wide vrition in the number of dynein rms nd rdil spokes seen in different xonemes within individul tissue smples, this study hs lso demonstrted the necessity for quntittive ssessment of lrge number of xonemes to chrcterize the entire smple. This cn be mde much simpler nd less tedious by enhncing ultrstructurl components using the fixtion techniques described in this pper. The increse in contrst chieved by the inclusion of tnnic cid nd MgS4 in fixtion procedures gretly increses the number of xonemes which re suitble for counting. The results of sttisticl nlyses between cili nd flgell Structurl Heterogeneity in Humn Axonemes 829

6 were similr whether the mens of individul smples were compred or the dt were pooled. In both cses there were significntly more microtubule doublets nd less centrl pir microtubules nd rdil spokes per xoneme in the cili thn in the sperm. It hs been previously suggested tht rdil spokes re more redily visulized in sperm tils (24, 25). It is difficult to postulte why this is so, but these differences should be recognized nd considered when drwing conclusions bout xoneml ultrstructure. A significntly greter number of sperm til xonemes hd n bnorml number of microtubule doublets compred with the cili (P <.2). This ws observed in 8:1 of the semen smples nd is further demonstrtion of the morphologicl heterogeneity of humn sperm in comprison with other species (26, 27). When bnormlities in the microtubule doublets were seen in semen smple, the number of doublets observed ws not consistently higher or lower thn the expected number of nine. In other words both nd might be present in the semen smple. This suggests tht the ssembly of tht prticulr xoneme my be defective nd tht the bnormlity is not due to either generl excess or deficiency of microtubules. The possibility ws considered tht the higher frequency of ssembly vritions in sperm til xonemes my be cused by difference in cell turnover rtes between the seminiferous nd nsl epitheli. However, t present, there is insufficient evidence to substntite this suggestion. When considering cili only, our results compre well with other studies of ultrstructure in norml tissue. Fox et l. (22) observed tht 4% of cili did not hve the microtubulr rrngement nd Rossmn et l. (15) found 1.7% of cili with n bnorml number of doublet microtubules nd.6% with bnorml number of centrl pir microtubules. We found 4.1 nd 1.3%, respectively. Clerly these vritions in the number of doublet nd centrl pir microtubules re cused by morphogenetic fctors ffecting the ssembly of microtubules during development ofthe xoneme rther thn problems of visulizing these structures with the fixtion nd stining procedures described. One notble feture of this study is the demonstrtion of reltively lrge proportion of xonemes with bnorml numbers of doublet microtubules in semen smple displying norml prmeters of 62% motile sperm nd motility index of 157. It might be expected tht the 2% of flgell which re bnorml re confined to the 38% of the sperm which re immotile. If the xoneml ultrstructure of such smple hd only been qulittively ssessed, it would certinly be described s bnorml. Interestingly, in the nsl biopsy of one helthy subject, Fox et l. (22) found tht 2% of cili hd bnormlities in the microtubulr orgniztion. Our dt clerly show tht simple "eyebll" observtion of few xonemes is not dequte nd tht comprehensive structurl ssessment must be combined with functionl studies. As indicted previously the subject from our study showed no such defects in his respirtory cili. This, in itself, is n importnt observtion prticulrly when considering studies tht describe norml cili nd bnorml flgell or vice vers (25, 28). This confirms other reports tht structurl defects of xonemes re not necessrily distributed throughout ll cell types nd my be confined to prticulr loction within the body. In some studies on xoneml ultrstructure (e.g., 24, 29), the Mrkhm rottion technique (3) hs been used to demonstrte the presence or bsence of dynein rms nd rdil spokes. The procedure involves rotting the xoneme through 4 degrees totl of nine times to superimpose the microtubules, dynein rms, nd rdil spokes before ssessment. For exmple, when only eight outer dynein rms re present, this method will incorrectly show nine outer dynein rms. Since our study hs shown tht the full complement of structures cn rrely be visulized, the rottion technique is not pproprite. This study hs clerly demonstrted tht vritions in xoneml ultrstructure occur frequently in norml functionl tissue. Despite our ttempts to mximize conditions for visulizing the vrious xoneml components, the idel xoneme with its full complement of dynein rms nd rdil spokes is rrely seen. We stress tht ssessment of xoneml ultrstructure must be quntittive nd well-documented, nd tht flse conclusions could esily be drwn if only few xonemes re qulittively observed nd the motility prmeters of the tissue not recorded. The dt provide not only structurl bse but lso functionl prmeters for both the respirtory epithelium nd sperm til. Such dt now provide definitive bse line for future studies of the role of ciliry nd sperm xoneml disorders in such diseses s bronchiectsis nd infertility. Acknowledgments The uthors grtefully cknowledge Mrs. Jo Hmer for ssessing motility prmeters of semen nd Professor W. J. Ewns of the Deprtment of Mthemticl Sttistics, Monsh University, for ssistnce with dt nlysis. This study ws supported by the Ntionl Helth nd Medicl Reserch Council of Austrli nd the Monsh University Specil Reserch Fund. References 1. Linck, R. W Advnces in the ultrstructurl nlysis of the sperm flgellr xoneme. In The Spermtozoon. D. W. Fwcett, nd J. M. Bedford, editors. Urbn & Schwrzenberg, Bltimore, MD Wrner, F. D Structure-function reltionships in cili nd flgell. In Electron Microscopy of Proteins. Vol. 1. Jmes R. Hrris, editor. Acdemic Press, London Cmner, P., B. A. Afzelius, R. Elisson, nd B. Mossberg Reltionship between bnormlities of humn sperm flgell nd respirtory trct disese. Int. J. Androl. 2: Pedersen, M., E. Morkssel, M. H. Neilsen, nd N. Mygind Krtgener's syndrome. Preliminry report on cili structure, function, nd upper irwy symptoms. Chest. 8: Afzelius, B. A A humn syndrome cused by immotile cili. Science (Wsh. DC). 193: Elisson, R., B. Mossberg, P. Cmner, nd B. A. Afzelius The immotile cili syndrome. A congenitl ciliry bnormlity s n etiologic fctor in chronic irwy infections nd mle sterility. N. Engi. J. Med. 297: Rossmn, C. M., J. B. Forrest, R. M. K. W. Lee, A. F. Newhouse, nd M. T. Newhouse The dyskinetic cili syndrome. Abnorml ciliry motility in ssocition with bnorml ciliry ultrstructure. Chest. 8: Pedersen, H., nd N. Mygind Absence of xoneml rms in nsl mucos cili in Krtgener's syndrome. Nture (Lond.). 262: Sturgess, J. M., J. Cho, J. Wong, N. Aspin, nd J. A. P. Turner Cili with defective rdil spokes: cuse of humn respirtory disese. N. Engl. J. Med. 3: Wilton, Teichthl, Temple-Smith, nd de Kretser

7 1. Cho, J., J. A. P. Turner, nd J. M. Sturgess Genetic heterogeneity of dynein-deficiency in cili from ptients with respirtory disese. Am. Rev. Respir. Dis. 126: Pedersen, M., nd N. Mygind Ciliry motility in the "immotile cili syndrome." First results of microphoto-oscillogrphic studies. Br. J. Dis. Chest. 74: Schneeberger, E. E., J. McCormck, H. J. Issenberg, S. R. Schuster, nd P. S. Gerld Heterogeneity of ciliry morphology in the immotile-cili syndrome in mn. J. Ultrstruct. Res. 73: Wite, D., R. Steele, I. Ross, J. St. J. Wkefield, J. Mcky, nd J. Wllce Cili nd sperm til bnormlities in Polynesin bronchiectsis. Lncet. II: Wite, D. A., J. St. J. Wkefield, J. B. Mcky, nd I. T. Ross Mucociliry trnsport nd ultrstructurl bnormlities in Polynesin bronchiectsis. Chest. 8: Rossmn, C. M., R. M. K. W. Lee, J. B. Forrest, nd M. T. Newhouse Nsl ciliry ultrstructure nd function in ptients with primry ciliry dyskinesi compred with tht in norml subjects nd in subjects with vrious respirtory diseses. Am. Rev. Respir. Dis. 129: Bum, G. L., Y. Roth, H. Teichthl, E. Ahronson, nd Z. Priel Ciliry bet frequency of respirtory mucosl cells: comprison of nsl nd trchel smpling sites. Am. Rev. Respir. Dis. 125(Suppl): Andersen, I., P. Cmner, P. L. Jensen, K. Philipson, nd D. F. Proctor A comprison of nsl nd trcheobronchil clernce. Arch. Environ. Helth. 29: Stnley, P., M. A. Greenstone, L. McWillim, nd P. J. Cole Nsl mucociliry clernce in ptients ttending nose clinic. Thorx. 38: Elisson, R Anlysis of semen. In Progress in Infertility, S. J. Behrmn, nd R. W. Kister, editors. Little, Brown nd Co., Boston Linck, R. W The structure of microtubules. Ann. NY Acd. Sci. 383: Wrner, F. D Ction-induced ttchment of ciliry dynein cross-bridges. J. Cell Biol. 77:R19-R Fox, B., T. B. Bull, nd G. B. Brden Vritions in the ultrstructure of humn nsl cili including bnormlities found in retinitis pigmentos. J. Clin. Pthol. 33: Aherne, W. A., nd M. S. Dunnill Morphometry. Edwrd Arnold Ltd., London Allen, R. D A re-investigtion of cross-sections of cili. J. Cell Biol. 37: Lungrell, G., L. Fonzi, nd A. G. Burrini Ultrstructurl bnormlities in respirtory cili nd sperm tils in ptient with Krtgener's syndrome. Ultrstruct. Pthol. 3: Fwcett, D. W The mmmlin spermtozoon. Dev. Biol. 44: Brtoov, B., F. Eltes, J. Lngsm, M. Snyder, nd J. Fisher Ultrstructurl studies in morphologicl ssessment of humn spermtozo. Int. J. Androl. Suppl. 5: Jonsson, M. S., J. R. McCormick, C. G. Gillies, nd B. Gondos Krtgener's syndrome with motile spermtozo. N.. Engl. J. Med. 37: Tksk, T., M. Sto, nd A. Onoder Atypicl cili of the humn nsl mucos. Ann. Otol. Rhinol. Lryngol. 89: Mrkhm, R., S. Frey, nd G. J. Hills Methods for the enhncement of imge detil nd ccentution of structure in electron microscopy. Virology. 2: Structurl Heterogeneity in Humn Axonemes 831