The oviductal cilia and Kartagener's syndrome*

Transcription

1 FERTllJTY AND STERILITY Copyright c 1986 The American Fertility Society Vol. 46, No.3, September 1986 Printed in U.8A. The oviductal cilia and Kartagener's syndrome* Peter McComb, M.B. t Lynn Langley, M.Sd Manuel Villalon, M.B.:!: Pedro Verdugo, M.D., Ph.D.:!: University of British Columbia, Vancouver, British Columbia, Canada, and University of Washington, Seattle, Washington Women wlw have Kartagener's syndrome (primary ciliary dyskinesia) mayor may not be fertile. The bronchial mucociliary clearance is reduced markedly in most of these women; this has led investigators to the conclusion that the cilia in the respiratory tract are immotile, and that "beating cilia may have no indispensable role in the female reproductive tract." Yet motile cilia are considered by many workers to be essential for normal ovum transport. More recently, bizarre ciliary motion has been described in the respiratory cilia of Kartagener's women. Our hypothesis was that the dyskinetic ciliary activity (or immotility) would be the same in both the respiratory and reproductive tracts and thus explain the fertility (or lack of it) in Kartagener's women. This report slwws an identical ultrastructure and absolute immotility of cilia in both the respiratory tract and reproductive tract of a woman with Kartagener's syndrome who has never conceived. From this concordance, we suggest that the fertility of Kartagener's women is explained by the dyskinetic motion of oviductal cilia, and that the ciliated endosalpinx is essential for human reproduction. Fertil Steril46:412, 1986 Women afflicted with Kartagener's syndrome (primary ciliary dyskinesia) have dextrocardia (Fig. 1), a variable degree of fertility, as well as bronchiectasis and sinusitis. 1, 2 The prevalence of this syndrome has been estimated at 1 in 40, One-half of those women with primary ciliary dyskinesia have situs inversus of the abdominal organs as well. In most such women the bronchial mucociliary clearance is reduced markedly. Veer- Received November 25, 1985; revised and accepted April 29, *Presented at the Fortieth Annual Meeting of The American Fertility Society, New Orleans, April 2 to 7,1984. treprint requests: Peter McComb, M.B., Department ofobstetrics and Gynaecology, Room 2H30, Grace Hospital, 4490 Oak Street, Vancouver, B.C., Canada V6H 3V5. tuniversity of Washington. 412 McComb et ai. Cilia and Kartagener's syndrome man and others have therefore concluded that the cilia in the respiratory tract of such women are immotile. 4 Yet in these same women fertility is often present, 5 leading some investigators to the conclusion that "beating cilia may have no indispensable role in the female reproductive tract."s Nevertheless, motile cilia in the mucosa of the fallo'pian tube have been considered by many workers to be essential for normal ovum transport The oviductal cilia are most plentiful in the distal ampulla and fimbria (57% of mucosal cells).l1 From the fimbria to the ampulla, to the isthmus, and to the interstitial tube, the proportion of ciliated cells decreases progressively. The direction of the metachronal ciliary beat is always prouterine in women. 12

2 Figure 1 Chest x-ray: dextrocardia. The individual cilium is a biologic unit that is common and fundamental to many diverse forms of life, such as the green alga Chlamydomonas 13 and to spermatozoa of numerous species. This phenomenon has allowed the study of ciliary physiology in lower species (viz the sperm of the sea urchin)14 with ready inference for human cilia. The ultrastructure of the cilium (Fig. 2)15 consists of nine doublets of "A" and "B" fibers that surround two central single microtubules. These central tubules originate from the basal plate of the cilium and rise to fuse at the ciliary tip. A sliding interaction between the dynein arms and the adjacent doublet allows the cilia to bend. The outer arms contain dynein, a high-molecularweight adenosine triphosphatase that couples the mechanochemical reaction believed to be the energy source for the ciliary beat. 16 With few exceptions, the ciliary ultrastructure is the same among different species and in each of the sites in the body where cilia are found. 4 Until now, the motility of the oviductal cilia has not been studied in a woman with Kartagener's syndrome. Turner and co-workers l7 have classified dyskinetic respiratory cilia into three categories, based upon the ultrastructural defect, viz I, dynein arm deficiency; II, radial spoke deficiency; and III, microtubular transposition. They suspected that complete ciliary immotility might not be present in each of the patients in their series Vol. 46, No.3, September 1986 because of the heterogeneous expression of disease. Several investigators l8-21 have now described ciliary motility of respiratory cilia despite proven ultrastructural defects within the cilia Pederson and Mygin 18 have suggested that a correlation might exist between specific anatomic defects (viz absent dynein arms). and degree and form of motility. However, it appears premature to define these associations. Nevertheless, "windscreen wiper" activity,21 "metronome" actions, "rotational movements" around the midpoint of the cilium, and "vibrational movements" characterized by the ciliary tip describing a "figure-of-eight" configuration20 have been observed directly in the respiratory cilia of up to 40% of all ciliated cells of women who would have been assigned previously to the "immotile cilia syndrome.,,19 If this same ciliary activity occurs in the oviductal cilia, as well as the respiratory cilia, then one would expect some women with the dyskinetic cilia syndrome to be fertile and others not, depending upon the degree and efficiency of the ciliary beat-conceivably such bizarre motility is enough to move the cumulus oophorus over the mucosal surfaces. This hypothetical model describes accurately the clinical finding of fertility in such women. 5, 6 Clearly, then, it remains only to be demonstrated that the motion (or lack of motion) of oviductal cilia is in accord with that of cilia elsewhere in the body. This final step in the unraveling of the apparent paradox of "fertile women with Kartagener's syndrome" is described below. MATERIALS AND METHODS The particular woman with Kartagener's syndrome that we chose to study had never con- Axoneme- Figure 2 A cilium in cross-section. McComb et ai. Cilia and Kartagener's syndrome 413

3 Figure 3 Laparoscopy. Left adnexum. F, fimbria; POF, preovulatory follicle. ceived. She had suffered from sinusitis, bronchiectasis, and was known to have situs inversus (Fig. 1). The infertility investigations included semen analysis, a postcoital test, hysterosalpingography, and assays of the serum thyroxine, follicle-stimulating hormone, luteinizing hormone, prolactin, dehydroepiandrosterone, estradiol, progesterone (P), and testosterone. All were within normal limits. Now aged 34 years, she had been attempting to conceive for 8 years. A laparoscopy was arranged to coincide with the time of ovulation, that is, when the ciliary ovum retrieval and transport occurs. The intraabdominal findings confirmed complete situs inversus, including the appendix, liver, and spleen. The genital. tract was morphologically normal; specifically, there was no evidence of previous pelvic inflammation. The left ovary contained a preovulatory follicle 2 cm in diameter (Fig. 3). Bronchial biopsy forceps (2 mm) were used to obtain representative tissues from each fimbriated end of the oviducts. The endometrium, the mucosa of the endocervix, and the epithelium of the nasal turbinates were also biopsied. For each site the tissues were prepared for immediate examination by phase contrast microscopy to assess any ciliary motility; the microscope was located within the operating room. Next, tissue cultures were prepared, grown, and replicated according to the method of Rumery et al. 22 to yield cultures rich in ciliated cells that are almost devoid -of secretory cells. The ciliated cells were grown and replicated in Rose chambers containing Eagle's medium (Steinberger modification) with 10% horse serum, ph 7.5, and incubated at 37 C. The immediate and later observations of any ciliary activity were conducted by direct micro414 McComb et al. Cilia and Kartagener's syndrome scopic observation (800-fold magnification) as well as by laser light-scattering spectroscopy. The cultured cells, as well as the tissue explants of ciliated cells, were equilibrated first in Hanks' solution, positioned in an inverted microscope modified for laser light scattering spectroscopy. The attenuated and collimated beam of He-Ne laser was directed at the culture chamber illuminating an area of approximately 8000 sq f.1m containing about 10,000 cilia. The laser light scattered by any moving cilia was collected by the objective lens of the microscope at an angle of 35 degrees from the plane of the chamber. Any changes in intensity of the scattered light would be detected as current fluctuations by a photomultiplier tube. The spectrum ofthe photo current fluctuations that gives directly the statistical distribution of frequencies of ciliary beat was processed on line by a fast Fourier transform digital spectrum analyzer developed in our laboratory.23 Tissue specimens for electron microscopy were fixed in half-strength Karnovsky's solution buffered in 0.1M cacodylate at ph 7.3, then postfixed in 1% OS04 in distilled water. Next, the tissues were dehydrated to 70% ethanol. Specimens for scanning electron microscopy were conditioned for critical point drying in 100% amyl acetate solution. A critical point bomb flushed the mucosae with liquid CO2 before critical point drying. The mucosal surfaces were mounted on aluminum pegs, sputter-coated with gold-palladium in a coater, and studied and photographed in a scanning electron microscope at 20 kv in the secondary electron mode. For transmission electron microscopy the specimens were taken from the 100% ethanol, placed in propylene oxide, and embedded in Epon. Sections were cut with a diamond knife at thicknesses of 600 to 1000 A, and stained with uranyl acetate and lead citrate, before study and photography in a transmission electron microscope. Blood was drawn for assay of serial P levels on the day preceding, the day of, and each of the 2 days after surgery. RESULTS Serum P levels (nanograms per milliliter) of 1 (day before surgery), 3 (day of surgery), and 7 (day after surgery) confirmed that the ovarian follicle seen at laparoscopy was imminently ovulatory. i I

4 DISCUSSION Figure 4 Transmission electron micrograph. Oviductal cilia. The arrow demonstrates partial outer dynein arm (original magnification, x 100,000). CILIARY MOTILITY-PHASE-CONTRAST MICROSCOPY Phase-contrast microscopy demonstrated conclusively that all the cilia in each of the biopsy sites were absolutely immotile. CILIARY MOTILITY-EARLY LASER SPECTROSCOPY The immediate observation by laser light-scattering spectroscopy demonstrated ciliary immotility. CILIARY MOTILITY-TISSUE CULTURE Normal cell development with formation of a confluent monolayer of ciliated' cells was observed.to take place after 6 days.of setting of tissue ex plants. However, despite optimal conditions, at no time :was ciliary movement seen. The laser-spec. troscopy technique reflected absolute immotility of the cilia in all tissues studied. -TRANSMISSION -ELECTRON MICROSCOPY We have demonstrated (1) that the structural and functional characteristics of oviductal cilia are the same as those of the cilia found elsewhere in the body in a woman with Kartagener's syndrome;.and (2) that the only cause for infertility identifiable in this woman with Kartagener's syndrome is the immotile cilia. The absolute immotility of the cilia observed in our study probably reflects full expression of this autosomally inherited syndrome. 16 The persistence of immotility, despite active cell -development and ciliary growth in tissue culture, is further proof of the intrinsic ciliary -defect, rather than any exogenous effect upon the cilia. Based on previous studies of tubal transport in the rabbit, we had:proposed that both myosalpingeal contraction and ciliary motility are sufficient-yet either alone is not-for the fallopian tube to retrieve the ovulated ovum and to then transport it through the oviduct at physiologically acceptable rates However, the ciliary immotility associated with infertility reported here supports the idea that, in humans, ciliary activity might not only be sufficient. but also necessary for normal ovum transport in the fallopian tube. The recent reports of bizarre ciliary motility in women with ciliary dyskinesia ' almost -certainly account for the observed fertility in some Kartagener's women. Our demonstration that the cilia look and behave the same in the oviduct as they do in other body sites:lends support to the thesis that the ciliary motility observed in the respiratory tract of many fertile.w omen with Kartagener's syndrome will also occur -within the fallopian tubes as well. The direct -observation of.oviductal ciliary. activity in fertile Kartagener's The major defect -revealed by the transmission electron microscope was partial or total absence of dynein :arms (Fig. 4). In some sections it was. possible to distinguish isolated outer arms, although in an incomplete form (Fig. 5). The central pair of singlets sometimes showed a pair of sym.metric armlike densities giving the appearance of a pair of spectacles. Supernumerary central singlets were often found..scanning ELECTRON MICROSCOPY Normal mucosa was seen with abundant ciliated and secretory cells. Although the cilia appeared very straight, their density was normal. Vol. 46, No. 3, September 1986 Figure 5 Transmission electron micrograph. Oviductal cilia. The arrow demonstrates arm-like projections-from central singlet (original magnification, x 200,000). McComb et al. Cilia and Kartagener's syndrome 415

5 women would strengthen our conclusion. This remains to be performed. That the only identifiable cause of the infertility in this patient is the ciliary immotilitypoints to the essential role the ciliated endosalpinx plays in reproduction. Acknowledgment. We express our appreciation to Richard Blandau, M.D., Ph.D., for his encouragement. REFERENCES 1. Kartagener M: Zur Pathogenese der Bronchiektasien. I. Bronchiektasien bei situs viscerum inversus. Beitr Klin Tuberk 83:489, Siewert AK: Uber einen Fall von Bronchiectasie bei einem Patienten mit Situs Inversus Viscerum. Bed Klin Wochenschr 41:139, Neffen H, Oehling A, Crisci CD: Kartagener's syndrome. Respiration 40:161, Veerman AJP, van Delden L, Feenstra L, Leene W: The immotile cilia syndrome: phase contrast light microscopy, scanning and transmission electron microscopy. Pediatrics 65:689, Afzelius BA, Camner P, Mossberg B: On the function of cilia in the female reproductive tract. Fertil Steril 29:72, Bleau G, Richer CoL, Bousquet D: Absence of dyne in arms in cilia of endocervical cells in a fertile woman. Fertil Steril 30:362, Blandau RJ: Comparative aspects of tubal anatomy and physiology as they relate to reconstructive procedures. J Reprod Med 21:7, Verdugo P, Blandau RJ, Tam PY, Halbert SA: Stochastic elements in the development of deterministic models of egg transport. In Ovum Transport and Fertility Regulation, Edited by MJK Harper, CJ Pauerstein, CE Adams, EM Coutinho, HB Croxatto, DM Paton. Copenhagen, Scriptor, 1976, p McComb P, Gomel V: The effect of segmental ampullary reversal on the subsequent fertility of the rabbit. Fertil Steril 31:83, Verdugo P, Lee WI, Blandau RJ, Halbert S, Tam P: Stochastic model for oviductal egg transport. Biophys J 29:257, Verhage HG, Bareither ML, Jaffe RC, Akbar M: Cyclic changes in ciliation, secretion and cell height of the oviductal epithelium in women. Am J Anat 156:505, Critoph FN, Denis KJ: Ciliary activity in the human oviduct. Br J Obstet Gynaecol 84:216, Witman GB, Plummer J, Sander G: Chlamydomonas flagellar mutants lacking radial spokes and central tubules: structure, composition, and function of specific axonemal components. J Cell BioI 76:729, Summers KE, Gibbons IR: Adenosine triphosphate-induced sliding of tubules in trypsin treated flagella of sea urchin sperm. Proc Natl Acad Sci USA 68:3092, Eliasson R, Mossberg B, Camner P, Afzelius B: The immotile cilia syndrome. N Engl J Med 297:1, Warner FD, Mitchell DR, Perkins CR: Structural conformation of the ciliary ATPase dynein. J Mol BioI 144:367, Turner JAP, Corkey CWB, Lee JYC, Levison H, Sturgess J: Clinical expressions of immotile cilia. Pediatrics 67:805, Pederson M, Mygin N: Ciliary motility in the immotile cilia syndrome. Br J Dis Chest 74:239, Rossman CM, Forrest JB, Lee RMKW, Newhouse MT: The dyskinetic cilia syndrome. Chest 78:4, Rossman CM, Forrest JB, Newhouse M: Motile cilia in immotile cilia syndrome. Lancet 1:1360, Rutland J, Cole P: Ciliary dyskinesia. Lancet 2:859, Rumery RE, Phinney E, Blandau RJ: Culture of Mammalian Embryonic Ovaries and Oviducts. In Methods in Mammalian Embryology, Edited by JC Daniel, Jr. San Francisco, W. H. Freeman & Co., 1970, p Verdugo P: Ca 2 + -dependent hormonal stimulation of ciliary activity. Nature 283:764, McComb et al. Cilia and Kartagener's syndrome