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Existence of a breaking point in cilia and flagella
J. theor. Biol. (1971) 33, 257-263
Existence of a Breaking Point in Cilia and Flagella J. J. BLUM Department of Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina 27706, U.S.A. (Received 11 January 1971)
Data in the literature indicate that the transitional region between the kinetosome and the flagellar or ciliary shaft of many Protozoan species is specialized as a breaking point. Only two Metazoan species have been examined from this point of view, and in both of them the transition region is also a breaking point. A similar phenomenon may occur in some sperm. Breakage occurs in response to a wide variety of chemical, osmotic, physical and mechanical treatments. Possible implications of this phenomenon are discussed. 1. Introduction It is the purpose of this paper to call attention to a hitherto unrecognized phenomenon, i.e. the existence of a region specialized for ease of breaking in cilia and flagella. The 9+2 axonemal complex typically found in cilia, flagella, and sperm tails is synthesized by the kinetosome. The details of this process are not known, but a universal feature of kinetosomal morphology is the presence of nine groups of three microtubules, referred to as abc (see Fig. I), and the absence of a central pair of microtubules (Stubblefield & Brinkley, 1967). At the region where the kinetosome ends and the flagellar shaft begins, the c subfiber in each of the nine groups of three microtubules terminates and the central pair begins. In view of the continuity of the a and b subfibers throughout the kinetosome and flagellar shaft, it seems a priori unlikely that the transition region between the kinetosome and the flagellar shaft should be a region specialized for easy breakage. In this paper 1 collect evidence scattered throughout the literature that the transition region is a breaking point in many cilia and flagella, as indicated by the remarkable variety of reagents which can induce the shedding of these organelles. 2. Ciliates The cilia of Tetrahymena can be stripped off by mild shearing forces after several different chemical treatments. Child (1959) discovered that exposure of Tetrahymena to 40% ethanol at - 10 “C followed by resuspension in T.B. 257 17
258
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FIG. 1. Diagram showing the structure of a cilium and kinetosome. (a) Structures seen in longitudinal section. (b) Transverse section of the ciliary shaft showing the typical 9 + 2 axonemal complex. (c) The arrangement of fibril triplets in the basal body. Note that the central pair of fibrils are absent in the kinetosome and, in cilia such as this, begin at the basal granule, which thus serves as a convenient marker for the transition region between the kinetosome and the ciliary shaft. In other types of cilia and flagella the details of the morphology of the transitional region are different, but in all cases the central pair of fibrils found in the ciliary shaft terminate at a region which marks the transition between cilium and kinetosome. This diagram is adapted from Sleigh (1962). More detailed pictures of the kinetosome and flagellar shaft are also given in Sleigh’s book.
0.1 M-KU and vigorous stirring would detach the cilia from the cells. He also found that treatment with 20 % glycerol at - 8 “C with occasional stirring for 5 to 10 min at 0 “C would result in deciliation. With either method the kinetosomes remained in the pellicle. Watson & Hopkins (1962) used an ethanol-ethylenediaminetetraacetic acid (EDTA) solution followed by the addition of excess calcium chloride to remove the cilia. Rosenbaum & Carlson (1969) modified this method, employing in sequence solutions containing EDTA, distilled water and CaCl,, followed by shear through a syringe needle. After deciliation, the cells regenerated their missing cilia, showing that the basal bodies had not been seriously damaged. Three groups of workers have devised methods for isolating the oral apparatus of Tetrahymena, which consists of four membranelles each comprising two or more rows of close packed basal bodies and their cilia (except for certain basal bodies of the undulating membrane which do not bear cilia). Whitson et al. (1966) lysed the cells by exposing them to a buffer saturated with indole, and collected the oral apparatus by centrifugation.
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POINT
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Williams & Zeuthen (1966) used 15 M-tertiary-butyl alcohol in conjunction with vigorous stirring, while Wolfe (1970) used a hypertonic medium followed by brief exposure to the detergent Triton X-100 and then vigorous resuspension in isolation medium without detergent. In each case the oral apparatus lost its cilia. Wolfe (1970) showed that in his procedure the break had occurred at the basal plate, where the ciliary axonemal fibers attach to the kinetosome, while according to Nilsson & Williams (1966) the cilia broke off just above the region where the central pair of fibers end. Tartar (1968) found that within two seconds after exposure to 20% sucrose the ciliate Stentor coeruleus began to shed its membranellar cilia, leaving the kinetosomes behind. The somatic cilia of members of the genus Paramecium are shed upon exposure to chloral hydrate, separation occurring at the junction of the cilium with the basal body (Kennedy & Brittingham, 1968). The basal body retains its terminal plate as well as its axosomal granule, so that all structures necessary for normal ciliary regeneration are present. It has also been reported that if paramecia are placed in a 0.02% solution of phosphacol [sic] the cilia begin to detach from the cell within 7 to 10 minutes (Seravin, 1961). During conjugation the ventral surfaces of Paramecium caudutum become apposed in a very precise manner. To permit close apposition and to allow for membrane fusion and fenestration the cilia “disappear”. According to Vivier & Andre (1961) this occurs by rupture at the base of the cilia, and, indeed, these authors comment that one can generally see free cilia around the mating couples. The chemical stimulus for this specific detachment process is unknown. It should be emphasized that this system is so far the only one where a clear physiological function for deciliation is indicated. 3. Flagellates Pringsheim (1956) referring to the flagella of euglenoid cells, states “as is well known, it breaks off under stress or irritation, in some species more, in others, less readily, and a new one is formed”. Euglena spirogyra, for example, cast off its flagellum from a point just distal to the flagellar swelling upon exposure to an irritant stimulus (Leedale, Meeuse & Pringsheim, 1965). Rosenbaum & Child (1967) state that “the flagella of Euglena gracih Z and of other flagellated protozoans can be amputated either by mechanical methods or by pH shock under conditions that more than 95% of the organisms in the culture regenerate new flagella.” Tibbs (1958) removed the flagella of Polytoma trvella by shaking the cells in distilled water containing a few drops of chloroform. Flagella can also be
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removed from Pofytoma by vigorously shaking the cells at -20 “C in a Tris-thioglycollate buffer containing about 60% glycerol (Brokaw, 1961). A variety of methods have been used to detach the flagella from Chlamydomonas.Mintz & Lewin (1954) found that treatment for 5 min with HCI at pH 3 would detach the flagella; the cells remained viable. Jones & Lewin (1960) removed the flagella simply by cooling cell suspensions to - 12 “C for 40 min. Hagen-Seyfferth (1959) deflagellated Chlamydomonasby brief exposure to dilute acetic acid or to high temperatures. Rosenbaum, Moulder & Ringo (1969) removed the flagella from Chfamydomonasmerely by exposure to mild shearing forces (1 min at 14,000 rev/min in a Virtis homogenizer). 98% of the flagella were removed without harming the cells, and the cells then formed new flagella. The flagella were detached just above the transitional region of the flagellar shaft and below the outer limit of the cell wall. Brokaw (1960) deflagellated Chlamydomonasby a glycerol procedure similar to that described above for Pofytomella. Finally, Marcus & Mayer (1963) found that the addition of dinitrophenol, salyrgan, or ATPT caused a rapid shedding of the flagella of Chlamydomonas. Simple mechanical agitation has been used by Dubnau (1961) and by Rosenbaum & Child (1967) to deflagellate the algae Uchromonas danica and by the latter to deflagellate Astasia longa and Euglena gracilis Z. 4. Metazoa
The tendency of cilia to break off at a point near the junction with the basal body is not confined to protozoan organelles. Auclair & Siegel (1966) induced deciliation of echinoderm blastulae by brief exposure of the embryo to sea water made hypertonic with NaCl, and the same method has been used to detach cilia from the gills of the mollusc Pecten irradians (Stephens & Linck, 1969). Iwaikawa (1967) found that addition of 1 ml of 1 M-sodium acetate to 1 ml of sedimented sea urchin embryos caused the cilia to detach within 30 seconds. He also reported [thus independently confirming Auclair & Siegel (1966)] that the use of hypertonic sea water would lead to ciliary detachment within 5 minutes, followed by regeneration, and that this process could be repeated up to five times. 5. Sperm
The tail (end piece) of a sperm is essentially a flagellum; its kinetosome is in the middle piece and may be located at the middle piece-end piece junction or more proximally within the middle piece. Some experiments suggest that i Chlun~~&dornonas also shed their flagella when carbonyl cyanide p-trifluoromethoxyphenylhydrazone is added to the medium (A. M. Mayer, personal communication).
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sperm may also have a breaking point at the middle piece-end piece junction. Austin (1968), for example, notes that sterility in certain bulls of the Guernsey breed of cattle is attributable to separation of sperm heads and tails; spermatogenesis is normal but, as the spermatozoa pass down the epididymis, heads and tails become detached, and the condition seems to affect all the spermatozoa. Electron microscopic investigation revealed that wherever the interior extremity of the detached tail could be identified, it could be seen to possess the proximal centride. In this case, therefore, breakage appears to occur behind the kinetosome, and this may not be comparable to the situations so far discussed, where breakage occurs at the junction of the flagellar shaft with the kinetosome. Further evidence on the possible existence of a breaking point in sperm comes from observations on sperm entry into eggs. Although in most metazoans the sperm tail enters the vitellus at fertilization (Austin, 19574, the sperm tail fails to enter the vitellus in normal fertilization in most eggs of the field vole (Austin, 19576) and in virtually all eggs of the Chinese hamster (Austin & Amoroso, 1959). In the polychaete worm Nereis both the middle piece and the flagellum are left outside (Wilson, 1928). Wilson (1895) also comments on fertilization in Toxopneustes that the head of the sperm (nucleus and middle piece) enters the egg and the tail is left outside the vitelline membrane. Thus the evidence concerning sperm suggests that the middle piece-end piece junction may be a breaking point in a few species. Systematic experiments based on the methods used to detach cilia and flagella would be needed to decide whether the existence of a breaking point in sperm is a general phenomenon. 6. Discussion
It is remarkable that in several species cilia or flagella are detached within seconds or a few minutes by such diverse procedures as heating, cooling, hypertonic sea water, dilute acid, sucrose, dinitrophenol, salyrgan, glycerol, ethanol and, in some cases, by mild shearing forces. For all the cases discovered (except sperm, where the situation is unclear) the break occurred in the immediate vicinity of the transitional region between the end of the kinetosome and the beginning of the flagellar shaft, and in every case (again excluding sperm) without appreciable damage to the kinetosome, at least as judged by their ability to regrow new cilia. Except for conjugation in paramecia, the physiological role of the breaking mechanism is obscure. It is possible to make an analogy with the process of autotomy in many species of Arthropoda and Reptilia. This
262
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J.
BLUM
process, whereby the animal is able to lose an appendage without seriously damaging the rest of the organism, has an obvious survival value, especially when coupled with the capacity for limb regeneration. On a far smaller scale cilia and flagella are appendages which could potentially endanger the cells to which they are attached. It is well known that cilia and flagella frequently stick to glass surfaces and similar sticking presumably may occur in natural environments. This problem was recognized by Calkins (1933), who wrote “With such energetic motile organs exerting a constant strain on the body there would seem to be some danger of their being pulled out, especially in those types with soft fluid bodies without firm periplasts”. Regardless of the teleological rationale for the existence of a breaking point in cilia and flagella, it appears on the basis of the evidence presented above that such breaking points do exist in many protozoan and in several metazoan species. The physiological role of this mechanism deserves further investigation, as does the molecular mechanism by which detachment is achieved. The author is the recipient of a Career Development (5 K3 GM 2341) from the National Institutes of Health.
Research Award
REFERENCES AUCLAIR, W. & SIEGEL, B. W. (1966). Science, N. Y. 154, 913. AUSTIN, C. R. (19574. Fertilization, p. 83. Englewood Cliffs, N.J.: Prentice Hall. AUSTIN, C. R. (19576). J. Amt. 91, 1. AUSTIN, C. R. (1968). Ultrastructure of Fertilization. New York: Holt, Rinehart & Winston. AUSTIN, C. R. & AMOROSO, E. C. (1959). Endeavour 18, 130. BROKAW, C. J. (1960). Expl Cell Res. 19, 430. BROKAW, C. J. (1961). Expl Cell Res. 22, 151. CALKINS, G. N. (1933). The Biology ofProtozoa, 2nd Ed., p. 143. Philadelphia: Lea & Febiger. CHILD, F. M. (1959). Expl Cell Res. 18, 258. DUBNAU, D. A. (1961). Ph.D. Thesis, Columbia University. HAGEN-SEYFFERTH, M. (1959). Planta 53, 376. IWAIKAWA, Y. (1967). Embryologia 9, 287. JONES, R. F. & LEWIN, R. A. (1960). Expl Cell Res. 19, 408. KENNEDY, J. R. & BRI~INGHAM, E. (1968). J. Ultrastruct. Res. 22, 530. LEEDALE, G. F., MEEUSE, B. J. D. & PRINGSHEIM, E. G. (1965). Arch. Mikrobiol. 50, 6s. MARCUS, M. & MAYER, A. M. (1963). In Microalgae and Photosynthetic Bacteria (special issue of PIant Cell Physiol., Tokyo), p. 85. MINTZ, R. H. & LEWIN, R. A. (1954). Cm. J. Microbial. 1, 65. NILSSON, J. R. & WILLIAMS, N. E. (1966). C. r. Trav. Lab. Curlsberg. 35, 119. PRINGSHEIM, E. G. (1956). Nova Acta Leopoldina 18, 1. ROSENBAUM, J. L. & CARLSON. K. (1969). J. Cell Biol. 40, 415. ROSENBAUM, J. L. & CHILD, F. M. (1967). J. Cell Biol. 34, 345. ROSENBAUM, J. L., MOULDER, J. E. & RINGO, D. L. (1969). J. Cell Biol. 41, 600. SERAVIN, L. N. (1961). Biokhimiya 26, 160. SLEIGH, M. A. (1962). The Biology of Cilia md Flagella. New York: Macmillan Co.
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STEPHENS, R. E. & LINCK, R. W. (1969). J. molec. Biol. 40, 497. STUBBLEFIELD. E. & BRINKLEY, B. R. (1967). In Formation and Fate of Cell Organelles. (K. B. Warren, ed.) p. 175. New York: Academic Press. TARTAR, V. (1968). Trans. Am. microsc. Sot. 87, 297. TIBBS, J. (1958). Biochim. biophys. acta 23, 636. VIVIER, E. & ANDRE, J. (1961). J. Protozool. 8, 416. WATSON, M. R. & HOPKINS, J. M. (1962). Expl. Cell Res. 28, 280. WHITSON, G. L., PADILLA, G. M., CANNING, R. E., CAMERON, I. L., ANDERSON, N. G. & ELROD, L. H. (1966). Nutn. Cancer Inst. Monogr. 21, 317. WILLIAMS, N. E. & ZEUTHEN, E. (1966). C. r. Trav. Lab. Carlsberg 35, 119. WILSON, E. B. (1895). An Atlas of the Fertikation and Karyokinesis of the Ovum. New York: Macmillan Co. WILSON, E. B. (1928). The Cell in Development and Heredity, 3rd ed. New York: Macmillan Co. WOLFE, J. (1970). J. Cell Sci. 6, 679.
Existence of a Breaking Point in Cilia and Flagella J. J. BLUM Department of Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina 27706, U.S.A. (Received 11 January 1971)
Data in the literature indicate that the transitional region between the kinetosome and the flagellar or ciliary shaft of many Protozoan species is specialized as a breaking point. Only two Metazoan species have been examined from this point of view, and in both of them the transition region is also a breaking point. A similar phenomenon may occur in some sperm. Breakage occurs in response to a wide variety of chemical, osmotic, physical and mechanical treatments. Possible implications of this phenomenon are discussed. 1. Introduction It is the purpose of this paper to call attention to a hitherto unrecognized phenomenon, i.e. the existence of a region specialized for ease of breaking in cilia and flagella. The 9+2 axonemal complex typically found in cilia, flagella, and sperm tails is synthesized by the kinetosome. The details of this process are not known, but a universal feature of kinetosomal morphology is the presence of nine groups of three microtubules, referred to as abc (see Fig. I), and the absence of a central pair of microtubules (Stubblefield & Brinkley, 1967). At the region where the kinetosome ends and the flagellar shaft begins, the c subfiber in each of the nine groups of three microtubules terminates and the central pair begins. In view of the continuity of the a and b subfibers throughout the kinetosome and flagellar shaft, it seems a priori unlikely that the transition region between the kinetosome and the flagellar shaft should be a region specialized for easy breakage. In this paper 1 collect evidence scattered throughout the literature that the transition region is a breaking point in many cilia and flagella, as indicated by the remarkable variety of reagents which can induce the shedding of these organelles. 2. Ciliates The cilia of Tetrahymena can be stripped off by mild shearing forces after several different chemical treatments. Child (1959) discovered that exposure of Tetrahymena to 40% ethanol at - 10 “C followed by resuspension in T.B. 257 17
258
I.
J.
BLUM
FIG. 1. Diagram showing the structure of a cilium and kinetosome. (a) Structures seen in longitudinal section. (b) Transverse section of the ciliary shaft showing the typical 9 + 2 axonemal complex. (c) The arrangement of fibril triplets in the basal body. Note that the central pair of fibrils are absent in the kinetosome and, in cilia such as this, begin at the basal granule, which thus serves as a convenient marker for the transition region between the kinetosome and the ciliary shaft. In other types of cilia and flagella the details of the morphology of the transitional region are different, but in all cases the central pair of fibrils found in the ciliary shaft terminate at a region which marks the transition between cilium and kinetosome. This diagram is adapted from Sleigh (1962). More detailed pictures of the kinetosome and flagellar shaft are also given in Sleigh’s book.
0.1 M-KU and vigorous stirring would detach the cilia from the cells. He also found that treatment with 20 % glycerol at - 8 “C with occasional stirring for 5 to 10 min at 0 “C would result in deciliation. With either method the kinetosomes remained in the pellicle. Watson & Hopkins (1962) used an ethanol-ethylenediaminetetraacetic acid (EDTA) solution followed by the addition of excess calcium chloride to remove the cilia. Rosenbaum & Carlson (1969) modified this method, employing in sequence solutions containing EDTA, distilled water and CaCl,, followed by shear through a syringe needle. After deciliation, the cells regenerated their missing cilia, showing that the basal bodies had not been seriously damaged. Three groups of workers have devised methods for isolating the oral apparatus of Tetrahymena, which consists of four membranelles each comprising two or more rows of close packed basal bodies and their cilia (except for certain basal bodies of the undulating membrane which do not bear cilia). Whitson et al. (1966) lysed the cells by exposing them to a buffer saturated with indole, and collected the oral apparatus by centrifugation.
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POINT
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Williams & Zeuthen (1966) used 15 M-tertiary-butyl alcohol in conjunction with vigorous stirring, while Wolfe (1970) used a hypertonic medium followed by brief exposure to the detergent Triton X-100 and then vigorous resuspension in isolation medium without detergent. In each case the oral apparatus lost its cilia. Wolfe (1970) showed that in his procedure the break had occurred at the basal plate, where the ciliary axonemal fibers attach to the kinetosome, while according to Nilsson & Williams (1966) the cilia broke off just above the region where the central pair of fibers end. Tartar (1968) found that within two seconds after exposure to 20% sucrose the ciliate Stentor coeruleus began to shed its membranellar cilia, leaving the kinetosomes behind. The somatic cilia of members of the genus Paramecium are shed upon exposure to chloral hydrate, separation occurring at the junction of the cilium with the basal body (Kennedy & Brittingham, 1968). The basal body retains its terminal plate as well as its axosomal granule, so that all structures necessary for normal ciliary regeneration are present. It has also been reported that if paramecia are placed in a 0.02% solution of phosphacol [sic] the cilia begin to detach from the cell within 7 to 10 minutes (Seravin, 1961). During conjugation the ventral surfaces of Paramecium caudutum become apposed in a very precise manner. To permit close apposition and to allow for membrane fusion and fenestration the cilia “disappear”. According to Vivier & Andre (1961) this occurs by rupture at the base of the cilia, and, indeed, these authors comment that one can generally see free cilia around the mating couples. The chemical stimulus for this specific detachment process is unknown. It should be emphasized that this system is so far the only one where a clear physiological function for deciliation is indicated. 3. Flagellates Pringsheim (1956) referring to the flagella of euglenoid cells, states “as is well known, it breaks off under stress or irritation, in some species more, in others, less readily, and a new one is formed”. Euglena spirogyra, for example, cast off its flagellum from a point just distal to the flagellar swelling upon exposure to an irritant stimulus (Leedale, Meeuse & Pringsheim, 1965). Rosenbaum & Child (1967) state that “the flagella of Euglena gracih Z and of other flagellated protozoans can be amputated either by mechanical methods or by pH shock under conditions that more than 95% of the organisms in the culture regenerate new flagella.” Tibbs (1958) removed the flagella of Polytoma trvella by shaking the cells in distilled water containing a few drops of chloroform. Flagella can also be
260
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removed from Pofytoma by vigorously shaking the cells at -20 “C in a Tris-thioglycollate buffer containing about 60% glycerol (Brokaw, 1961). A variety of methods have been used to detach the flagella from Chlamydomonas.Mintz & Lewin (1954) found that treatment for 5 min with HCI at pH 3 would detach the flagella; the cells remained viable. Jones & Lewin (1960) removed the flagella simply by cooling cell suspensions to - 12 “C for 40 min. Hagen-Seyfferth (1959) deflagellated Chlamydomonasby brief exposure to dilute acetic acid or to high temperatures. Rosenbaum, Moulder & Ringo (1969) removed the flagella from Chfamydomonasmerely by exposure to mild shearing forces (1 min at 14,000 rev/min in a Virtis homogenizer). 98% of the flagella were removed without harming the cells, and the cells then formed new flagella. The flagella were detached just above the transitional region of the flagellar shaft and below the outer limit of the cell wall. Brokaw (1960) deflagellated Chlamydomonasby a glycerol procedure similar to that described above for Pofytomella. Finally, Marcus & Mayer (1963) found that the addition of dinitrophenol, salyrgan, or ATPT caused a rapid shedding of the flagella of Chlamydomonas. Simple mechanical agitation has been used by Dubnau (1961) and by Rosenbaum & Child (1967) to deflagellate the algae Uchromonas danica and by the latter to deflagellate Astasia longa and Euglena gracilis Z. 4. Metazoa
The tendency of cilia to break off at a point near the junction with the basal body is not confined to protozoan organelles. Auclair & Siegel (1966) induced deciliation of echinoderm blastulae by brief exposure of the embryo to sea water made hypertonic with NaCl, and the same method has been used to detach cilia from the gills of the mollusc Pecten irradians (Stephens & Linck, 1969). Iwaikawa (1967) found that addition of 1 ml of 1 M-sodium acetate to 1 ml of sedimented sea urchin embryos caused the cilia to detach within 30 seconds. He also reported [thus independently confirming Auclair & Siegel (1966)] that the use of hypertonic sea water would lead to ciliary detachment within 5 minutes, followed by regeneration, and that this process could be repeated up to five times. 5. Sperm
The tail (end piece) of a sperm is essentially a flagellum; its kinetosome is in the middle piece and may be located at the middle piece-end piece junction or more proximally within the middle piece. Some experiments suggest that i Chlun~~&dornonas also shed their flagella when carbonyl cyanide p-trifluoromethoxyphenylhydrazone is added to the medium (A. M. Mayer, personal communication).
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POINT
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sperm may also have a breaking point at the middle piece-end piece junction. Austin (1968), for example, notes that sterility in certain bulls of the Guernsey breed of cattle is attributable to separation of sperm heads and tails; spermatogenesis is normal but, as the spermatozoa pass down the epididymis, heads and tails become detached, and the condition seems to affect all the spermatozoa. Electron microscopic investigation revealed that wherever the interior extremity of the detached tail could be identified, it could be seen to possess the proximal centride. In this case, therefore, breakage appears to occur behind the kinetosome, and this may not be comparable to the situations so far discussed, where breakage occurs at the junction of the flagellar shaft with the kinetosome. Further evidence on the possible existence of a breaking point in sperm comes from observations on sperm entry into eggs. Although in most metazoans the sperm tail enters the vitellus at fertilization (Austin, 19574, the sperm tail fails to enter the vitellus in normal fertilization in most eggs of the field vole (Austin, 19576) and in virtually all eggs of the Chinese hamster (Austin & Amoroso, 1959). In the polychaete worm Nereis both the middle piece and the flagellum are left outside (Wilson, 1928). Wilson (1895) also comments on fertilization in Toxopneustes that the head of the sperm (nucleus and middle piece) enters the egg and the tail is left outside the vitelline membrane. Thus the evidence concerning sperm suggests that the middle piece-end piece junction may be a breaking point in a few species. Systematic experiments based on the methods used to detach cilia and flagella would be needed to decide whether the existence of a breaking point in sperm is a general phenomenon. 6. Discussion
It is remarkable that in several species cilia or flagella are detached within seconds or a few minutes by such diverse procedures as heating, cooling, hypertonic sea water, dilute acid, sucrose, dinitrophenol, salyrgan, glycerol, ethanol and, in some cases, by mild shearing forces. For all the cases discovered (except sperm, where the situation is unclear) the break occurred in the immediate vicinity of the transitional region between the end of the kinetosome and the beginning of the flagellar shaft, and in every case (again excluding sperm) without appreciable damage to the kinetosome, at least as judged by their ability to regrow new cilia. Except for conjugation in paramecia, the physiological role of the breaking mechanism is obscure. It is possible to make an analogy with the process of autotomy in many species of Arthropoda and Reptilia. This
262
J.
J.
BLUM
process, whereby the animal is able to lose an appendage without seriously damaging the rest of the organism, has an obvious survival value, especially when coupled with the capacity for limb regeneration. On a far smaller scale cilia and flagella are appendages which could potentially endanger the cells to which they are attached. It is well known that cilia and flagella frequently stick to glass surfaces and similar sticking presumably may occur in natural environments. This problem was recognized by Calkins (1933), who wrote “With such energetic motile organs exerting a constant strain on the body there would seem to be some danger of their being pulled out, especially in those types with soft fluid bodies without firm periplasts”. Regardless of the teleological rationale for the existence of a breaking point in cilia and flagella, it appears on the basis of the evidence presented above that such breaking points do exist in many protozoan and in several metazoan species. The physiological role of this mechanism deserves further investigation, as does the molecular mechanism by which detachment is achieved. The author is the recipient of a Career Development (5 K3 GM 2341) from the National Institutes of Health.
Research Award
REFERENCES AUCLAIR, W. & SIEGEL, B. W. (1966). Science, N. Y. 154, 913. AUSTIN, C. R. (19574. Fertilization, p. 83. Englewood Cliffs, N.J.: Prentice Hall. AUSTIN, C. R. (19576). J. Amt. 91, 1. AUSTIN, C. R. (1968). Ultrastructure of Fertilization. New York: Holt, Rinehart & Winston. AUSTIN, C. R. & AMOROSO, E. C. (1959). Endeavour 18, 130. BROKAW, C. J. (1960). Expl Cell Res. 19, 430. BROKAW, C. J. (1961). Expl Cell Res. 22, 151. CALKINS, G. N. (1933). The Biology ofProtozoa, 2nd Ed., p. 143. Philadelphia: Lea & Febiger. CHILD, F. M. (1959). Expl Cell Res. 18, 258. DUBNAU, D. A. (1961). Ph.D. Thesis, Columbia University. HAGEN-SEYFFERTH, M. (1959). Planta 53, 376. IWAIKAWA, Y. (1967). Embryologia 9, 287. JONES, R. F. & LEWIN, R. A. (1960). Expl Cell Res. 19, 408. KENNEDY, J. R. & BRI~INGHAM, E. (1968). J. Ultrastruct. Res. 22, 530. LEEDALE, G. F., MEEUSE, B. J. D. & PRINGSHEIM, E. G. (1965). Arch. Mikrobiol. 50, 6s. MARCUS, M. & MAYER, A. M. (1963). In Microalgae and Photosynthetic Bacteria (special issue of PIant Cell Physiol., Tokyo), p. 85. MINTZ, R. H. & LEWIN, R. A. (1954). Cm. J. Microbial. 1, 65. NILSSON, J. R. & WILLIAMS, N. E. (1966). C. r. Trav. Lab. Curlsberg. 35, 119. PRINGSHEIM, E. G. (1956). Nova Acta Leopoldina 18, 1. ROSENBAUM, J. L. & CARLSON. K. (1969). J. Cell Biol. 40, 415. ROSENBAUM, J. L. & CHILD, F. M. (1967). J. Cell Biol. 34, 345. ROSENBAUM, J. L., MOULDER, J. E. & RINGO, D. L. (1969). J. Cell Biol. 41, 600. SERAVIN, L. N. (1961). Biokhimiya 26, 160. SLEIGH, M. A. (1962). The Biology of Cilia md Flagella. New York: Macmillan Co.
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