Insect Sperm Cells

Insect Sperm Cells BACCIO BACCETTI Institute of Zoology. University of Siena. Italy I. I1. 111.

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Introduction . . . . . . . . . . . . . . . . . The Cell Surface . . . . . . . . . . . . . . . . The Acrosomal Complex . . . . . . . . . . . . . A. The Typical Triple-layered Insect Acrosomal Complex . B. The Bilayered Acrosomal Complex. . . . . . . . . C. The Acrosomal Complex with Only Two Outer Layers . D. Monolayered Acrosomal Complex . . . . . . . . E. Total Absence of Acrosomal Complex . . . . . . . The Nucleus . . . . . . . . . . . . . . . . . A . Nuclear Shape . . . . . . . . . . . . . . . B. Submicroscopic Structure . . . . . . . . . . . C . Chemical Characteristics . . . . . . . . . . . D. Physical Characteristics . . . . . . . . . . . . The Centriolar Region . . . . . . . . . . . . . . A . The Centriole . . . . . . . . . . . . . . . B. The Centriole Adjunct . . . . . . . . . . . . C. The Initial Segment of the Axoneme . . . . . . . . The Axial Flagellar Filament or Axoneme . . . . . . . A. The Microtubules . . . . . . . . . . . . . B. The Central Sheath . . . . . . . . . . . . . C. The Link-heads . . . . . . . . . . . . . . D. The Coarse Fibres . . . . . . . . . . . . . E . The Axonemal Matrix . . . . . . . . . . . . Mitochondria . . . . . . . . . . . . . . . . . A. Normal Mitochondria . . . . . . . . . . . . B. Mitochondria Transformed into Derivatives with a Crystalline core . . . . . . . . . . . . . . . . . C. Absence of Mitochondria . . . . . . . . . . . Accessory Ordered Flagellar Bodies . . . . . . . . . . A . Structured Bodies Flanking Normal Mitochondria . . . B. Structured Bodies Flanking the Mitochondria1 Derivatives with a Crystalline Matrix . . . . . . . . . . . C. Structured Bodies Replacing Mitochondria . . . . . 315

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Spermatozoa Possessing a Double Flagellar Apparatus or Being Devoid of it. . . . . . . . . . . . . . . . . . A. The Paired Spermatozoa . . . . . . . . . . . B. SpermatozoaPossessing Two Axonemes . . . . . . C. Non-flagellate Spermatozoa . . . . . . . . . . X. Motility. . . . . . . . . . . . . . . . . . . A. Motile Mechanisms . . . . . . . . . . . . . B. Metabolic Aspects of Motion . . . . . . . . . . C. The Problem of Sperm Capacitation . . . . . . . XI. SpermatozoaPolymorphism and Genetics . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . IX.

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I. INTRODUCTION

Among the animal phyla that have adapted themselves permanently to land life, the spermatozoon had to undergo thorough changes in its structure, metabolism and motility as a consequence of the onset of internal fertilization. The aquatic environment having been abandoned, spermatozoa were required to swim in a completely different medium, which was produced usually by the two partners at fertilization. Annelida, Mollusca, Arthropoda and the vertebrates have separately, but sometimes with amazing analogies, devised new forms of male gametes. In some arthropod groups still breeding in water, a “primitive” sperm model is retained which might be referred to as “aquatic” in type, e.g. in the Merostomata (AndrC, 1965; Baccetti, 1970; Shoger and Brown, 1970) and in the crustaceans Mystacocarida (Brown and Metz, 1967), Cirripedia and Branchiura (Brown, 1966, 1970). The outstanding features of this “aquatic” sperm type can be found in the most ancient representatives of almost all phyla, including Amphioxus among the Chordata (Baccetti et al., 1972f). These “primitive” features are represented by a roundish head, a great number of normal mitochondria rich in cristae and a relatively short flagellum with a plain axial filament simply organized according to the 9 + 2 pattern. This model was inherited by all arthropod classes, including those which are now definitely terrestrial. If allowance is made for some slight variations, this model is retained by primitive Arachnida, the scorpions (AndrC, 1963); among primitive Myriapoda, by the Pauropoda, Symphyla (Rosati et al., 1970) and Chilopoda (Horstmann, 1968); as well as by the Collembola and Diplura among primitive insects (Dallai, 1967, 1970; Baccetti et al., 1972d).

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When we follow the pathway of their evolution as revealed by extant forms, it may be observed that the Arachnida, Myriapoda and Insecta have subsequently refined their spermatozoa. Most Arachnida and Myriapoda produce “encysted” sperms (Baccetti, 1970) which are subsequently released in the female genital tract. All the spiders exhibit a peculiar 9 + 3 flagellum (Baccetti et al., 1970d; Reger, 1970) which is apparently of little functional significance because the encysted sperm is transported passively. As a consequence, the most evolved of the two lines, Opilionids (Reger, 1969) and Acarina (Reger, 1963; Breucker and Horstmann, 1968) on one side, and high Diplopoda (Reger and Cooper, 1968; Horstmann and Breucker, 1969a, b; Reger, 1971) on the other, have non-flagellate spermatozoa. Conversely, the spermatozoon of insects has evolved in the direction of increased motility: its shape, structure, metabolism and locomotory capabilities are tremendously differentiated. In many instances they attain an extreme specialization which is related to: (1 ) the mode of sperm transmission (internal fertilization always being the case); (2) the duration of its survival in the male genital tract or within spermatophores and spermathecas; (3) the type of fluid they have to swim in; and (4) the complexity of the egg envelopes they must penetrate. Typical features of the insect spermatozoon (Fig. 4) are the following: a generally very slim shape with an extremely elongated head; a bi- or three-layered acrosomal complex; an exceedingly long tail whose axial filament is flanked by accessory structures usually derived from mitochondria1 transformations. Along these lines evolutionary changes have resulted in a highly diversified and, in some respects, puzzling picture. Since some insight into the structure and function of spermatozoa has been gained during the past few years, it seems worth while to determine how far our knowledge on this subject has progressed. 11. THE CELL SURFACE

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Mature insect spermatozoa are surrounded by the typical triple-layered membrane which appears markedly asymmetrical in electron microscopical sections (Fig. 9) due to the fact that with all fixation and staining procedures its outer electron opaque layer consistently is as thick as the intermediate light (30 A ) and opaque

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inner (20 A) layers put together. According to Baccetti et al. (1 97 la) both opaque layers show acid phosphatase activity (Fig. 1C). They appear smooth after freeze-etching whereas the intermediate transparent layer contains a number of scattered globules (Fig. IA). The outer layer reacts positively to all the methods for glycoproteins, which suggests that this membrane resembles a common plasma membrane with a strongly thickened glycocalyx. This model in which, moving from the inside to the outside, the layers are consecutively some 20, 30 and 60 A in thickness and the glycocalyx appears structureless, is shared by the vast majority of insect spermatozoa. It was referred to as the “fruit-fly type membrane” by Baccetti et al. (1 97 1a). In addition, three progressively more elaborate arrangements are known. The first one (Fig. 1) was described in Aphaniptera and given the name of “flea type membrane” again by Baccetti et al. (1971a). In this instance, the outer sperm wall is about 200 A thick, since a 130 A thick glycocalyx is attached to the normal plasma membrane which averages 70 A . The glycocalyx has a peculiar structure. It is arranged around the spermatozoon in transverse striae, with a periodicity of some 140 A , overlying the outer surface of the plasma membrane (Fig. IB). A more complex arrangement (Fig. 2) was found among locusts (“locust type membrane”) by Baccetti et al. (1971a). Here, as was described in earlier observations (Roth, 1957; Kessel, 1967), the sperm wall is as thick as 400 A, due to a glycocalyx exceeding 300 A in thickness (Fig. 2A) and comparable to the fuzzy coat of many protozoa. This coat is made up of rodlets 300 A long and 70 A wide inserted almost perpendicularly into the sperm surface. They are arranged in tetrads in which every rodlet is 2 0 A apart from its neighbour (Fig. 2B). Each tetrad is about 70 A away from the others. Both the striated envelope of the Aphaniptera spermatozoon and the fuzzy one in locusts are derived from an abundant amorphous glycoprotein material coating the spermatid. This material becomes organized over the plasma membrane during sperm maturation. More elaborate and less obvious is the origin of the most complex type of sheath so far known, namely that of Lepidoptera, which was described in butterflies by AndrC (1 959, 1962) and Phillips (1 970b), and in moths by Riemann (1970). This type, however, has been recently confined to the eupyrene sperms (Phillips, 197 1). This sheath (Fig. 3A) is made up of numerous appendages, called lacinate

Fig. 1. The cell periphery of the spermatozoon of Ctenocephalus canis. (From Baccetti et aZ., 1971a.) (A) Frozen-etched preparation, showing the striation of the outer surface (s) and the granulated (g) space of the cell membrane. ~60,000.(B) Longitudinal section of the plasma membrane (p) and of the array of fibres striating the cell coat (s). ~150,000.(C) Acid phosphatase on plasma membrane (p) and on the outer cell coats (s). ~80,000.

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Fig. 2. The cell periphery in the spermatozoon of Aiolopus strepens. (From Baccetti et al., 1971a.) (A) Cross section of mature sperms, showing the brush-structured outer coat (c) surrounding the plasma membrane (p). ~120,000.(B) Negative staining of the fragmented cell membrane, showing the frontal view of the rodlets arranged in tetrads (s). ~360,000.

Fig. 3. The lacinate coat of the Lepidopteran spermatozoon. (From Phillips, 1971.) (A) Transverse section of eupyrene testicular spermatozoa showing the radiate outer appendages. x 120,000. (B) Transverse section of eupyrene spermatozoa after entering the female. The radial appendages are substituted by two layers of extracellular material (arrows). x 106,000.

Fig. 3 AIP-14

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appendages by Andre (1959, 1962), radially arranged all around the spermatozoon. Each appendage consists of many juxtaposed laminae 90 A apart from one another. One of these appendages is lattice-like instead of laminar and lies at the level of doublet 1 of the flagellum. This crown of appendages seems to be derived from a peculiar amorphous small body referred to as “light band” by Yasuzumi and Oura (1964). In the spermatids, this body arises externally and differentiates into the radially arranged laminar structures. The glycocalyx is not the only structure to grow and develop during the last phases of spermiogenesis. There are also striking changes in the plasma membrane associated with the change in cell shape which occurs when the roundish spermatid turns into an impressively elongated spermatozoon. In the mature spermatid, progressively more complex membrane systems develop. These are flat cisternae which envelope the main organelles, notably the axial filament and mitochondria, demarcating the form of the future sperm and excluding wide regions of the spermatid cytoplasm which will later be shed. These membranes were regarded as endoplasmic reticulum by Yasuzumi et al. (1958) as well as by Ito (1 960). It was pointed out by Baccetti et al. (1 972b) that they are derived from the Golgi complex, and that during sperm maturation they usually fuse with the limiting membrane. In some places they simply adhere to the inner surface of the plasma membrane, doubling its thickness. Elsewhere a total fusion takes place, eventually resulting in a simple plasma membrane; again at other sites the original plasma membrane is eliminated and replaced by a newly formed membrane of Golgi origin. The morphological significance of these membranes, which clearly originate from the Golgi complex, is still debated. In spermatocytes, Sakai and Shigenaga (1 967) favour the view that the tubular endoplasmic reticulum originates from the Golgi. It may perhaps be preferable (Baccetti et al., 1972b) to call them Golgiderived membranes, mindful of the fact that many of them are going to build the definitive limiting membrane. Alteration of the membranes does not stop at the end of spermatogenesis, but undergoes further modifications during sperm transfer from the male to the female. The elaborate lacinate appendages of the eupyrene spermatozoa in the Lepidoptera undergo breakdown in the male genital duct. The material they are composed of is rearranged into concentric bands encircling the formerly naked apyrene sperms (Phillips, 1971). Once they have reached the female (Fig. 3B), both eupyrene and apyrene sperms possess identical

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Fig. 4. Schematic drawing of conventional models of a mature insect spermatozoon. The acrosome region is the three-layered one of Aiolopus (Orthoptera), the centriole region the radiated one of Bacillus (Phasmoidea), the tail the most complete known, of Tenebrio (Coleoptera): A, acrosome; AB, accessory bodies; AC, centriole adjunct; AT, accessory tubules; CC, central cylinder; CF, coarse fibres; CS, central sheath; CT, central tubules; D, doublets; DD, dissociated doublets; IC, inner cone; EL, extraacrosomal layer; GM, Golgi derived membranes; LH, link-heads; MD, mitochondria1 derivatives; N, nucleus: OC, outer cylinder; PM, plasma membrane; RL, radial links; RLA, radial laminae.

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envelopes. In moths, these envelopes differentiate into two parts having different paracrystalline structures (Riemann and Thorson, 1971). On the contrary, the “fuzzy coat” of the locust sperm is completely digested in the female genital tract (Renieri and Vegni, 1972). 111. THE ACROSOMAL COMPLEX

A multi-layered acrosomal complex is present among the most primitive insects such as the Diplura, Collembola and Thysanura (Dallai, 1970, 1972; Baccetti et al., 1972d). There are but small increases in complexity of this basic arrangement up to the most evolved Pterygota, like the Coleoptera (Baccetti et al., 1972a) and Hymenoptera (Hoage and Kessel, 1968). For these latter examples, therefore, it is possible to propose a general model of acrosomal structure for the whole class. The various modifications exhibited by single orders and species can be derived from this model and always consist of various structural simplifications. In extreme cases, this organelle is completely absent. The insect acrosome, as is the rule for all animals, is typically derived from the Golgi complex (the earliest evidence in this regard was provided by Beams et al. (1 956), Clayton et al. (1 958), Ito (1 960), Gatenby and Tahmisian (1 959) and others). A. THE TYPICAL TRIPLE-LAYERED INSECT ACROSOMAL COMPLEX

This arrangement, with few variations, is present in almost all the Pterygota. It has been described in great detail for Orthoptera and Blattoidea (Kaye, 1962; Eddleman et al., 1970; Baccetti et al., 1971c), in Mecoptera (Baccetti et al., 1969c), in Aphaniptera (Baccetti, 1968), in the coleopteran Tenebrio (Baccetti et al., 1972a) and in the honey-bee (Hoage and Kessel, 1968). Generally, three juxtaposed layers (sometimes described as concentric) are encountered on the inside of the plasma membrane (Fig. 5A). The outermost “extraacrosomal layer” is an aggregation of granular cytoplasmic material which is concentrated between the plasma membrane and the acrosome proper during spermiogenesis. It occurs in a€l the instances mentioned here, but is particularly abundant in the more primitive orthopterans (Kaye, 1962; Shay and Biesele, 1968; Baccetti et al., 1971c). Its significance is utterly obscure. Beneath it the acrosome proper is found. This layer originates from the proacrosomal granule of the spermatid which is synthesized by

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Fig. 5. Three different arrangements of the acrosome complex in insects. (A) The three-layered model of Tenebrio: a, acrosome; el, extraacrosomal layer; ic, inner cone; pm, plasma membrane; n, nucleus. ~60,000. (From Baccetti el al., 1972a.) (B) The bilayered model of Campodea: a, acrosome; ic, inner cone. ~48,000.(From Baccetti and Dallai, 1972.) (C) The monolayered model of Chloeon: a, acrosome; n, nucleus. ~60,000 (From Baccetti et aZ., 1969b.)

the Golgi apparatus. Synthesis is most frequently associated with the concave side of its cisternae (Gatenby and Tahmisian, 1959; Kaye, 1962; Phillips, 1966a). In a single case it was seen to arise from the convex side (Phillips, 1970b). Histochemical investigations (Zylberberg, 1969; Baccetti et al., 1971c) have shown that the acrosome consists of a rather stable glycoprotein which is resistant to extraction. By negative staining the acrosome reveals a 95 vertical period and a 25 A horizontal period (Warner, 1971). The acrosome is

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entirely surrounded by its own triple-layered membrane, which previously encircled the proacrosomal granules derived from the Golgi (Kaye, 1962), and contains acid phosphatase (Baccetti et al., 1972b). Within the acrosome proper the third layer is encountered. This may be rodlike or conical, in the latter case being called “inner cone”. This is a compact structure, which is formed in the spermatid in an interspace between the acrosomal granule and the nuclear membrane, independently of the acrosome proper or of the Golgi region (Kaye, 1962). The characteristic interstitial membrane, which in the spermatid forms between the nuclear membrane and the proacrosomal granule (Kaye, 1962), is seen to break down at the site where this inner rodlet arises. Generally, no trace of the interstitial membrane is left in the mature sperm. The inner rodlet is endowed with histochemical properties diverging considerably from those of the acrosome; it consists of a protein core surrounded by a glycoprotein halo (Baccetti et al., 197 1 c). Information as to its origin and significance is entirely lacking. According to its position and ontogenesis, it might be regarded as comparable to the vertebrate perforatorium, which also contains carbohydrates (Sandoz, 1970), but its chemical nature is unknown. So far, the functional interpretation of the different components of the insect acrosomal complex is difficult to determine since even the mammalian one is as yet poorly understood. In mammals, according to Austin (1948) and Wada et al. (19S6), the acrosome contains hyaluronidase, while the perforatorium is believed to contain lysine (see Dan, 1967). Among the insects, the acrosomal hyaluronidase is completely absent (Baccetti et al., 197 1c). However it is not known whether lysines are similarly concentrated in the acrosome. This triple-layered acrosomal arrangement is generally conical in shape. However, in the tettigonioid Orthoptera it is arrow-like, and its stratification is also detectable inside the arrow limbs (Phillips, 1970b; Baccetti et al., 1 9 7 1 ~ ) In . the spermatozoon it generally lies apically to the nucleus. B. THE BILAYERED ACROSOMAL COMPLEX

This arrangement is very similar to the triple-layered acrosomal complex described above, the only difference is the absence of the outer extraacrosomal layer (Fig. SB). This arrangement is typical of the Apterygota. The extraacrosomal layer is consistently lacking in

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Collembola (Dallai, 1970), Protura, Diplura (Baccetti et al., 1972d) and Thysanura (Dallai, 1972). In this respect the bilayered arrangement might be regarded as more primitive than the triple-layered one, rather than the reverse. But, pending clarification of the nature and function of the extraacrosomal layer, nothing can be said about it at present.

C. THE ACROSOMAL COMPLEX WITH ONLY TWO OUTER LAYERS

This is a modification of the triple-layer arrangement; it consists of the acrosome proper and the extraacrosomal layer. Examples of this arrangement are found in phasmoidea (Baccetti et al., 1972b) and among a number of Homoptera (Folliot and Maillet, 1970). When the acrosome of Hemiptera has reached a considerable length, it becomes enriched by inner and outer microtubules which maintain its rigidity (Payne, 1966; Tandler and Moriber, 1966; Folliot and Maillet, 1970) and are still present in the mature spermatozoon. Since the inner rodlet is found in the more primitive arthropods and in the apterygotes as well, this arrangement might be interpreted as an involution of the triple-layered one. In Bacillus, glucose-6phosphatase is detectable in a restricted basal zone (Baccetti et al., 1972b) which might indicate a specialized area, although any speculation in this regard is far from easy.

D. MONOLAYERED ACROSOMAL COMPLEX

This structure consists of the acrosome alone (Fig. SC), enveloped by the acrosomal membrane and the plasma membrane. It has been reported in many groups: it occurs regularly in Ephemeroptera (Baccetti et al., 1969b), in Plecoptera (Baccetti et al., 1970e), in many Heteroptera (Barker and Riess, 1966; Herold and Munz, 1967; Mazzini, 1970), in Corrodentia , Mallophaga and Thysanoptera (Baccetti et al., 1969d), in those Trychoptera which possess the acrosome (Phillips, 1970b), in Lepidoptera (Phillips, 1971), in Diptera (Bairati and Perotti, 1970; Perotti, 1969; Tates, 1971, in Drosophila; Phillips, 1966b in Sciara) and in the Psychodidae (Baccetti et al., 1972c). This type of acrosome is always of very small size, and is often displaced to a lateral position with respect to the nucleus.

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E. TOTAL ABSENCE OF THE ACROSOMAL COMPLEX

This is occasionally found in some orders in which certain species possess an acrosome. Some instances are found in some of the more highly evolved species of the Isoptera (Baccetti et al., 1972e); in Coccoidea among Rhynchota, (Robison, 1966); in some species among Trychoptera (Baccetti et al., 1970e; Phillips, 1970b), in Oryctes among Coleoptera (Furieri, 1963a). In a whole order, Neuroptera (Baccetti et al., 1969c), the acrosome seems to be consistently lacking. This condition seems also due to involution. All the information summarized above seems to suggest that the triple-layered arrangement, typical of the pterygotes, was derived from a primitive bilayered acrosome model (rodlet and acrosome sensu stricto) as still retained by Apterygota. In some of these forms the acrosome may lose its rodlet and become bilayered (but the two outer layers are retained, hence not corresponding to the two found in Apterygota), monolayered or disappear altogether. The acrosomal layer itself is the only one present whatever the arrangement. IV. THE NUCLEUS

In a recent review on the sperm nucleus, Chevailler (1 970) points out that it is usually given less attention than the other sperm organelles, possibly due to its structural homogeneity and the uniformity of its transformations during spermiogenesis in various species. However, the survey carried out by this author brings into focus many important features, which will be discussed in the following section. A. NUCLEAR SHAPE

Among the insects, the sperm nucleus is as a rule fairly elongated, spindle-shaped anteriorly and truncated posteriorly. It is considerably compact, occasionally helicoidally arranged (Fig. 6b), sometimes even coiling around the flagellum as in the Mecoptera or Thysanura (Baccetti et al., 1969c, d). In Dahlbominus (Hymenoptera) some sperms have the helix twisted to the right, others to the left (Wilkes and Lee, 1965). Only in the spermatozoa provided with two axial filaments does the nucleus display a short flat shape as found in Anoplura and Mallophaga (Baccetti et al., 1969d). Aberrant

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non-flagellate sperms found in the most highly evolved Isoptera have a spheroidal or flattened nucleus (Baccetti e t al., 1972e). B. SUBMICROSCOPIC STRUCTURE

As a rule, in the mature insect sperm the nucleus appears compact and homogeneous in electron micrographs. This structure is attained during spermiogenesis. The chromatin in the young spermatid contains fibrils some 30-40 thick (Yasuzumi and Ishida, 1957; Gibbons and Bradfield, 1957; Dass and Ris, 1958; Gall and Bjork, 1958; Nebel, 1957), which are rapidly converted into thicker fibrils (1 00-200 arranged longitudinally (Werner, 1966; Chevaillier, 1970). These fibres then fuse into laminae which sooner or later coalesce. In the Apterygota the laminar pattern is also retained by the nearly mature spermatozoon (Bawa, 1964; Dallai, 1967) and the nucleus becomes compact just before its expulsion. In some instances, the nucleus instead of being homogeneous, exhibits a honey-comb texture (Yasuzumi and Ishida, 1957). The nuclear material does not undergo uniform morphological changes during spermiogenesis. Sometimes the central zone is the first to condense, at other times it is the periphery (Chevaillier, 1970). Among Psyllidae (Le Menn, 1966) only half the nucleus is occupied by chromatin, even in the mature sperm. During spermiogenesis the nuclear envelope is surrounded by a layer of microtubules which function in the compression and elongation of the nucleus, and which will disappear in the mature sperm (see, in particular, Kessel (1966, 1967)). DNA is seen to aggregate preferentially at the microtubule level (Baccetti e t al., 1972b) as is the case in most animals (Ferraguti and Lanzavecchia, 1971). Wide portions of the nuclear membrane are seen to form blebs which are pinched off into vesicles and dispersed into the cytoplasm (Kessel, 1970). Pores progressively decrease in number until they are no longer evident in the mature sperm. In some instances, however, the nucleus seems to take part in the elaboration of substances which are retained in the sperm cytoplasm. it was suggested by Werner (1 966) that these nuclear derivatives may give rise to the centriole adjunct and similar structures. However, this finding calls for further verification. Stable microtubules may also be endonuclear and protrude outwards in evaginations of the nuclear and plasma membrane. This was pointed out in the Neuroptera (Baccetti et al., 196%). This condition is quite common in the

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Fig. 6 . Two different forms of insect sperm nuclei. (A) The conical model of Bacillus, with an inner cavity containing polyribosome-like granules. ~ 9 0 , 0 0 0(From Baccetti et al, 1972b.) (B) The coiled arrangement found inMachi1is.x 90,000. (From Dallai, 1972.)

star-shaped nucleus of certain crustaceans (Decapoda), in which motile arms are found (Pochon-Masson, 1965, 1968a, b; Yasuzumi and Lee, 1966; Eliakova and Goriachkina, 1966;Anderson and Ellis, 1967),but is exceptional among insects. The scale insects, whose sperm is highly aberrant (see below), also seem to belong to this category.

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In the lower Arthropods, and in general among sea-dwelling invertebrates, the existence of a canal running within the nucleus, housing processes that are connected with acrosomal structures, is of common occurrence. As mentioned earlier, the sperm nucleus in insects is usually compact. However, in Bacillus, Baccetti and his collaborators (1 972b) describe a cribriform structure (Fig. 6A) full of polyribosomes positioned in an extranuclear space in the centre of the nucleus. C. CHEMICAL CHARACTERISTICS

It is well known that in the mature spermatozoon the nucleus consists essentially of the haploid DNA content. Atypical lines may lack DNA, as in apyrene lepidopteran sperms (Meves, 1903). In some instances, e.g. some carabids, polyploid sperms have been described (Bouix, 1963). Conversely, no RNA is present. In two insects, Philaenus and the house cricket, extrusion of ribonucleoprotein granules through the nuclear pores during the spermatid stage has been documented (Maillet and Gouranton, 1965; Kaye and McMaster Kaye, 1966). Along with DNA, proteins are contained in the sperm nucleus. These are mainly basic and consist of basic amino acids which replace the histones occurring in the nucleus of normal somatic cells and spermatogonias. Protamines, essentially consisting of arginine, lysine and histidine, chiefly studied in fish, are the most frequent. Other insects (Orthoptera and Drosophila) have, on the contrary, a category of histones particularly rich in arginine as a basic nuclear protein, which are synthesized in the cytoplasm during spermiogenesis (Das et al., 1964), using messenger RNA produced prior to meiosis (Bloch and Brack, 1964; Claypool and Bloch, 1967). Protamines and arginine-rich histones are essential for the compact nucleus of the mature spermatozoon. It was reported by Shoup (1967) that a particular Drosophila mutant being unable to achieve the transition from histones to arginine-rich histones possesses sterile sperms, with non-condensed nuclei. D. PHYSICAL CHARACTERISTICS

The important problem concerning the organization of material within the sperm nucleus, is still unresolved. Since X-ray observations in insects are not available, all information has been obtained from polarized light, fluorescence and electron microscopical investiga-

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tions. In other groups of animals we know that the nucleoprotein chains in the sperm nucleus are variably arranged, that is, ranging from an almost parallel array to haphazardly distributed chains. Working on the same material in insects, where the sperm nucleus is always exceedingly long and narrow, InouC and Sat0 (1 962, 1966) and MacInnes and Uretz (1968) came to opposite conclusions concerning Orthopteran spermatozoa. According t o the former workers, deoxyribonucleoproteins are organized into two superhelices of 8000 each, resulting from the coiling of a 1500 helix which consists in its turn of slender bundles (200 According to the latter workers, however, no superhelices exist and the DNA lies in arrays parallel t o the major axis of the sperm. The second interpretation has been substantially confirmed by Zirwer et al. (1970). Chevaillier (1 970), in an effort to resolve the controversy, proposes a scheme in which the chromosomes are oriented end-to-end along the length of the sperm nucleus. In this connection, Chevaillier mentions that Hughes-Schrader ( 1946) has reported a constant arrangement of the haploid chromosome set in Coccidae and quotes Taylor’s (1 964) work on Orthoptera, which by step-wise labelling with thymidine has revealed different levels of DNA in the sperm nucleus corresponding to a linear arrangement of chromosomes in the head.

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V. THE CENTRIOLAR REGION A. THE CENTRIOLE

In insect spermatids the normal orthogonal orientation of the two centrioles has been observed occasionally (Breland et al., 1966). More often either a single centriole is found (Friedlander and Wahrman, 1966), or two, as in biflagellate sperms (Fig. 7). However, the fine structure of this organelle has been established by only a few workers (Hoage and Kessel, 1968; Anderson and AndrC, 1968; Phillips, 1970b). They have the classical type of nine helicoidally arranged triplets (AndrC and Bernhard, 1964; Fawcett, 1966), comparable to those recently described by Ross (1968). Nevertheless, a more obscure problem today is the persistence of a centriole in the mature insect spermatozoon. As a matter of fact, no electron micrographs so far published show a classical centriole in a fully mature sperm and the presence of this organelle as late as the completion of spermiogenesis was denied by Phillips (1970b) in a

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Fig. 7. The centrioles (c) of the biflagellate sperm of Haemtopinus suis Both are . Baccetti el aZ., 1970a.) surrounded by the adjunct centriole (ca). ~ 6 0 , 0 0 0(From

recent discussion of this problem. At the same time, a centriole-like organelle was accurately described by Perotti ( 1970) in Drosophila sperm. However, in this case all the C tubules of the triplets are continuous with the accessory flagellar fibres (which, as will be reported presently, have a different origin) and the central pair of tubules is also present. These profiles could more easily be interpreted as the tip of the normal axial filament, where the accessory fibres may simulate triplets since they lie close to the peripheral doublets. At present, therefore, it may be assumed that a centriole does not occur in the mature insect sperm. B. THE CENTRIOLE ADJUNCT

This structure is a compact, basophilic sleeve, already known from light microscopy, characteristic of the spermatozoon in almost all insects. Called by various terms (“centriole adjunct”, which was

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coined by Gatenby and Tahmisian in 1959, is used most often), this organelle has been described in a variety of species in almost every order (see Cantacuzene’s survey (1 970)). It is generally of granular appearance (Fig. 7), with granules ranging between 160 and 320 a;it is fibrous in a few cases, e.g. in the house cricket and in Sciara (Kaye and McMaster-Kaye, 1966; Phillips, 1966a). Generally, it is more abundant in young spermatids (over 1 p 2 across) than in older ones where it becomes more compact (Breland et al., 1966). It is even more reduced in spermatozoa, where in some instances it may not be found (Phillips, 1970b). The origin of the centriole adjunct is still unknown. Werner (1965) favours the view of a nuclear origin starting from some material derived from a particular porous caudal portion of the nuclear membrane. Cantacuzhe (1970) showed that its synthesis was induced by the spermatid centriole. The aberrant multiplication of the centrioles is paralleled by a corresponding multiplication of the centriolar adjunct. Transitory contacts with Golgi cisternae and the endoplasmic reticulum are not sufficient to secure a supply of material by these organelles. Thus, the origin of the centriole adjunct is still uncertain. It seems to be responsible for the disappearance of the centriole around which it forms. Only recently has the nature of the centriolar adjunct been clarified by histochemical methods (Baccetti et al., 1969c; Yasuzumi et al., 1970; Gassner, 1970) and autoradiography (Baccetti et aZ., 1970a). There is little doubt that it consists of a ribonucleoprotein. This demonstration enabled Yasuzumi and his collaborators to propose a possible relationship between centriole adjunct and the chromatoid body of the spermatid. The ribonucleoprotein nature of the chromatoid body has long been claimed in many animals (Sud, 1961a, b; Daoust and Clermont, 1955) as well as in insects (Tandler and Moriber, 1965). But Eddy’s (1 970) recent demonstration that the chromatoid body is devoid of ribonucleoproteins, and that it arises not from the nucleus but rather from the interstices between the mitochondria (Fawcett et al., 1970) nullifies the main support to the derivation: nucleus-chromatoid body-centriole adjunct. The function of the centriole adjunct is not clear. Beginning with Gatenby and Tahmisian (1959) and Gatenby (1 96 l), most workers have always favoured a mechanical function for this compact collar, in the sense of it fastening the sperm head and tail together. This view is shared by Breland et al. (1966) and Fawcett and Phillips (1 969). Only after the more recent chemical analyses have other

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hypotheses been advanced, regarding it as a deposit of basic histones released by the nucleus during its enrichment with arginine-rich histones (Cantacuzkne, 1970) or as a concentration point of substances to be utilized in the assembly of the flagellar filaments (Yasuzumi et al., 1970). Since the initial segment of the axial filament is housed inside the centriole adjunct which apparently functions to initiate the movement of the flagellum (Baccetti et al., 1972b), a mechanical action of the centriole adjunct appears more and more probable. C. THE INITIAL SEGMENT OF THE AXONEME

Granted the absence of the centriole in insect spermatozoa, it follows that the first tubular units one encounters directly behind the sperm head are the fibres of the axial flagellar complex. In some instances, the accessory tubules are the first t o appear, in others the doublets; in all cases, the two central units arise shortly thereafter. This initial segment of the axoneme fits into ‘a corresponding nuclear indentation, or inside the sleeve formed by the condensed centriole adjunct. Its structure is complicated by the fact that it contains some units which are no longer found in the following tracts. The most widespread arrangement (Fig. 8C) consists of two short concentrically orientated cylinders connected by nine longitudinal radial laminae dividing the interspace into sectors (Fig. 4). Each doublet and each accessory tubule are contained in one sector, lying against a

Fig. 8. The “initiation motor” region of insect sperms. (A) and (B) Two different levels inDrosophiZa, showing cross links (arrows)between the opposite doublets. x 120,000. (From Perotti, 1970.) (C) Cross section in Euccillus, showing the outer ( 0 ) and the inner (i) cylinder and the radial laminae (I). ~90, 000.(From Baccetti et aZ., 1972b.)

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lamina. The two central units are contained within the central cylinder. Tail sections in different functional moments show that laminae and cylinders lie at variable angles and the laminae themselves differ in their extension. These laminae are strongly reactive for ATPase activity. Baccetti and his collaborators ( 1972b) have recently proposed that this complex may function as a device to trigger the rotation of the inner cylinder within the outer one, thereby dragging the heads of the axoneme tubules into this motion, so as to initiate a wave which then propagates along the fibres (Section X A). An arrangement of this type is evident morphologically in Phasmoidea, Orthoptera, Dermaptera and Neuroptera. In the spermatozoa of Drosophilu, the two cylinders are not present in the neck region (Perotti, 1970), but cross links are evident between the opposed triplets (doublets plus accessory fibres) each embedded in a dense sheath (Fig. 8A, B). In the coleopteran Tenebrio, both the inner cylinder and the radial or cross laminae (or strands) are missing, while a thick outer cylinder is found, which connects the heads of the outer fibre crown one after the other. In point of fact, the wave pattern in the three arrangements is considerably different (see Section X A). The organization of the axoneme is undoubtedly very complex and is relevant to the general problem of flagellar motion. In a schematic diagram of a normal cilium at different levels, Gibbons and Grimstone (1 960) and Holwill(l966) illustrate a structure composed of two cylinders and radial laminae in proximity to the head, followed by another consisting of a single outer cylinder and one more with cross strands. As a working hypothesis, it can be assumed that from the primitive multiphase mechanism occurring in cilia one stage or another was preferentially developed by spermatozoa, resulting in the formation of different kinds of waves. VI. THE AXIAL FLAGELLAR FILAMENT OR AXONEME

By slightly modifying Warner’s definition (1970), the axoneme (Fig. 9) may be regarded as the whole of the flagella complex limited by the outermost crown of microtubules. Hence, the classical Fig. 9(A) Cross section of sperm tails of Ceratitis capitata (Diptera): a,axoneme; m, mitochondrial derivatives; p, plasma membrane. ~120,000.(B) Cross section of the

sperm tail of Tenebrio molitor (Coleoptera): ab, accessory bodies; at, accessory tubules; cf, coarse fibres; ct, central tubules; d, doublets; Ih,link heads; md, mitochondrial derivatives. ~240,000.(From Baccetti et aZ., 1972a.)

Fig. 9

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microtubules, arranged according to the fundamental 9 + 2 or 9 + 9 + 2 patterns, along with the elements connecting them with one another contribute to its formation, while both mitochondria and membranes are excluded. A. THE MICROTUBULES

In the vast majority of insects, the axoneme consists of two central tubules, nine doublets and nine accessory tubules (Figs 4,9B, 10A, D, E). This scheme was first brought to light by Rothschild (1955) in the honey-bee and by Yasuzumi (1956) in Drosophila. It was then established that most insect orders belong to this category. The only exceptions are the Protura (Fig. 12A), which have a bizarre axoneme with 12 (Acerentulus) or 14 (Acerentomon) doublets and nothing else (Baccetti et al., 1972d), and the other primitive Apterygota (Collembola and Japigidae), in which the accessory tubules are missing and the basic 9 + 2 pattern, typical of classical primitive sperms, is retained (Krzystofowicz and Byczkowska-Smyk, 1966; Dallai, 1967, 1970; Baccetti and Dallai, 1972). Accessory tubules are also lacking (Fig. 10B) in the two kindred orders Mecoptera (Baccetti et al., 1969c) and Aphaniptera (Baccetti, 1968; Phillips, 1969), as well as in two species of Trichoptera (Phillips, 1970b) and in Thysanoptera (Baccetti et al., 1969d). Other arrangements have occasionally been reported, e.g. the Ephemeroptera (Fig. 1OC) lack both central tubules, thus being 9 + 9 + 0 (Baccetti et al., 1969b; Phillips, 1969); the same is true of a Psocid, which however has a solid central fibre (Phillips, 1969). Culicid Diptera, on the contrary, have only one central tubule and are thus 9 + 9 + 1 (Breland et al., 1966). A non-identified mycetophilid dipteran has three central tubules, hence is 9 + 9 + 3 (Phillips, 1970b). The two trichopteran species mentioned above have as many as seven central tubules being therefore 9 + 7 (Phillips, 1969). The dipteran Sciara coprophila (Fig. 11) lacks central tubules, having instead 70 doublets and 70 spirally arranged accessory tubules in its huge flagellum (Makielski, 1966; Phillips, 1966b). In general, therefore, there are three kinds of tubules in the axoneme of insect spermatozoa: accessory, doublets and central tubules. These are most often present in the 9 + 9 + 2 pattern. Differences among the various tubule categories have already emerged from structural studies (Andre, 1961; Baccetti and Bairati, 1964) and were conclusively demonstrated by Behnke and Forer

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Fig. 10. Cross section of five different sperm tails: ab, accessory bodies; at, accessory tubules; ct, central tubules; d, doublets; md, mitochondria1 derivatives. (A) Neuronia 9 + 9 + 2 (Trichoptera). ~130,000. (From Phillips, 1970.) (B) Panorpa annexa, 9 + 2 (Mecoptera) ~ 9 0 , 0 0 0 .(From Baccetti et al., 1969c.) (C) Chloeon dipteron, 9 + 9 + 0 (Ephemeroptera). ~120,000.(From Baccetti et al., 1969b.) (D)Noronectuglauca, 9 + 9 + 2 (Rhynchota). ~90,000. (E) Chrysopa curnea 9 + 9 + 2 (Neuroptera). ~90,000.(From Baccetti e t al., 1969c.)

Fig. 11. Cross sections of the sperm tail of Sciuru coprophila. ~45,000.(From Phillips, 1966b.)

( 1967) by means of differential enzymatic extractions upon ultrathin

sections. The following knowledge is now available about this subject.

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34 1

1. Central Tubules

a. Tubules. The diameter of central tubules from different axonemes

varies from 200 to 300 A. Their wall is about 70-80 A and its globular structure was first demonstrated by Bairati and Baccetti (1965). After dissociation it resolves into subunits appearing as fibrils made up of an alignment of globules or microcylinders, some 40 A thick. As a rule, the wall of each tubule consists of about 10 subunits. In various mammals, AndrC and ThiCry (1963) and Pease (1963) counted 10 of them, but among insects their number seems a little higher, i.e. 13 (Fig. 9B), according to the measurements of Phillips (1966), which were carried out on many species from different orders. There are 10-12 according to Danilova (1 969) in Bombyx mori. It is important to recall that 12-13 subunits are found in cytoplasmic microtubules and in those of the flagellum of protozoa (Ledbetter and Porter, 1964; Gall, 1965, 1966; Behnke and Zelander, 1967; Ringo, 1967; Fuge, 1968). In insects, no detailed chemical investigation has been performed'on the tubule wall. It is,

however, known to be proteinaceous (Behnke and Forer, 1967);

possibly the tubulin of Mohri (1968). The latter is an actin-like protein with a 6 s sedimentation constant and molecular weight of about 120,000 changing to about 60,000 after denaturation. It contains a guanine nucleotide and binding sites for colchicine (Shelanski and Taylor, 1968). The above protein was studied in echinoderm sperm (Plowman and Nelson, 1962; Nelson, 1966; Shelanski and Taylor, 1968) as well as in the doublets of Tetrahymena cilia (Stevens et al., 1967; Renaud et aZ., 1968). The biochemical unit (mol. wt = 120,000) is likely to correspond to two of the 40 a microcylinders described by morphologists (Shelanski and Taylor, 1968). According to Fine (1971) it seems to be composed of two different subunits, with mol. wt. = 56,000 and 54,500 respectively, with different amino acid composition (Bryan and Wilson, 1971). These subunits would then be able to aggregate into longitudinal fibrils or to follow a helical course. This explains the different appearance shown by the same microtubules when examined longitudinally after dissociation in negative stain (Danilova, 19691, while in cross section they always look the same (Thomas, 1970; Henley, 1970). The central tubules may be hollow (Fig. 10B, E), as in Mecoptera, Plecoptera and Neuroptera (Baccetti et aZ., 1969c, 1970e) or may contain a globular central core (Fig. 9), of the same size as the wall subunits, as is the case among Diptera (Bairati and Baccetti, 1965; Phillips, 1966b; Perotti, 1969; Warner, 1970). In some cases, they may be filled with microcylinders, some

342

B. BACCETTI

Fig. 12(A) Cross section of the 14 + 0 axoneme of Acerentomon majus (Protura). ~80,000. (From Baccetti et al., 1972d.) (B) Cross section of the 9 + 9 + 2 sperms of Campodeu (Diplura) showing the disordered migration of accessory tubules near the mitochondrion (arrows). ~48,000.(From Baccetti and Dallai, 1972.) (C)A more caudal section of the 9 + 9 + 2 sperm of Campodea (Diplura) showing the ordered disposition of the nine accessory tubules (arrows) near the mitochondrion. ~75,000.(From Baccetti and Dallai, 1972.) (D) The tip of the sperm tail in Telamona spec. (Rhynchota). The doublets appear dissociated. ~62,000.(From Phillips, 1970a.)

40 A in diameter, about eight per tubule, as in Gryllus (Kaye, 1964, 1970). Very often (in Trichoptera, Lepidoptera, Phasmoidea), however, the two tubules (Fig. 14A, B, D) become filled not only by protein, but also by a glycogen-like polysaccharide (Baccetti et a l , 1969a, 1970e, 1972b). b. The projections. Short side-projections (Kessel, 1967) are seen to jut out from the tubule wall, 150-170 A apart from one another and helically coiled (Fig. 13B, C) around the tubules (Perotti, 1969). In the cilia and flagella of Protozoa these projections exist in two series in tubule I and in one series in tubule I1 (Hopkins, 1970), or are present only in tubule I (Chasey, 1969). They seem to be separated from the tubule wall and vertically interconnected by filaments (Chasey, 1969).

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According to Warner (1970) they might be the binding site of the tubule wall to the “central sheath” which is also helically coiled around both structures with a similar pitch of 160 A . The central tubules do not generally undergo transformations towards the tail end, where they usually terminate after the accessory tubules and shortly before the doublets. In a few instances, e.g. Drosophila and the Trichoptera (Perotti, 1969; Baccetti et al., 1970e) they end after the doublets. Rarely, as in Tenebrio, they are the first to disappear (Baccetti et al., 1972a). In Gryllotalpa they are filled with a glycoprotein which becomes tanned, thereby making the distal half of the tail rigid and stiff (Baccetti et al., 197 1b). 2. Doublet tubules

a. The tubules. These are the only structures which are consistently

present in all types of flagellate spermatozoa. Doublets are, as a rule, nine in number, lying at some distance from one another. They can be oriented and numbered from 1 to 9 .according to the general pattern of cilia and flagella (Afzelius, 1959; Gibbons and Grimstone, 1960): so that doublet 1 lies on the median plane normal to the line linking the two central tubules. Doublet 2 follows in the arm direction and so on. Only Protura exhibit 12 or 14 doublets (Baccetti et al., 1972d); in Sciara there are 70 (Phillips, 1966a: Fig. 11). In cross section both tubules in each doublet (Fig. 9B) are found to consist of a B tubule (200-230 A ) slightly larger than the A tubule (200 A). From tubule A two arms are seen to extend. The outer one is about 35 mp long and directed towards the flagellum centre, the inner one is only 20 mp long. These measurements have been checked by Allen (1968) in Tetrahymena cilia and seem to be valid for the insect flagellum (Warner, 1970). The arm pairs, up to 100 in thickness, are found every 200-220 A along the tubule length (Phillips, 1970b). The doublet wall and the arms are not made up of the same material. Both are proteinaceous (Fig. 14C) but, like the central tubules, the doublet consists essentially of tubulin (although this evidence concerns protozoan cilia and sperms from Echinodermata as emerges from studies carried out primarily by Renaud et al. in 1968 and by Shelanski and Taylor in the same year). The arms, however, consist of dynein, a 14s protein unit with a molecular weight of 600,000, arranged in linear polymers endowed with ATPase activity (Fig. 14E). These observations are based on studies of Tetrahymena and the sea urchin by Gibbons (1963) and Gibbons and Rowe (1 965). It is most unlikely that the sperm doublets differ

a

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Fig. 13

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345

from this structure. Unlike the tubule walls, the arms of the A tubules are rich in ATPase activity (Baccetti et al., 1972b). Both consist of globular units of comparable size with those of tubulin. However, they differ from one another in features other than their size. Firstly, as shown by Phillips ( 1 9 6 6 ~ )tubule ~ B is not a real tubule, but a groove adhering to tubule A. Nevertheless, the subunit number seems to be consistently 13 and it may be supposed that B opens against A. Furthermore, a chemical difference between the two tubules has emerged from enzymatic extractions of various types as reported by Behnke and Forer (1967), though in both cases they are proteins (Baccetti et al., 1970e). As a rule, tubule A appears “solid” and tubule B “hollow”. When, as in Gryllus, both are filled up with globular units, these are in greater number in A than in B, though the latter is narrower (Kaye, 1970). In Bacillus spermatids tubule B has glucose-6-phosphatase activity (Baccetti et al., 1972b). A new technique of heat fractionation devised by Stephens (1970) separated the doublets of sea urchin spermatozoa into two units. He was thereby able to isolate A and B tubules which had a similar guanine nucleotide content, but different levels of amino acids, in particular cystein. This explains why only tubule A can bind to dynein and raises some questions about other microtubule categories. The contractility problem also remains open since, again in the sea urchin, tubulin does not possess antigenic properties like those of actin (Stephens, 1970). Recently, Behnke et al. (1971) were able to demonstrate in Nephrotoma sperm tails, bundles of 15-20 actin filaments independent of the axoneme tubules. This actin may be involved in sperm motility, but its exact localization in the sperm is still unknown. The doublets are usually the longest sperm tubules and are the last to terminate at the tail tip. Often they dissociate into their two units (Phillips, 1 9 6 6 ~ ) In . the Homoptera the tail splits into four strands (Fig. 12D), each containing some doublets, which eventually dissociate (Folliot, 1970). In Gryllotalpa the doublets are invaded in Fig. 13. Fine structural pattern of the insect axoneme. (A) Radial links (RL) and peripheral doublet (D) of Drosophila, negatively stained. The doublet (D) appears not dissociated. ~ 140,000.(From Bairati and Perotti, 1970.) (B) Central tubule of Drosophila, negatively stained. Projections spaced at 170 A are evident. ~200,000.(From Bairati and Perotti, 1970.) (C) Platinum shadowed central tubule of Aiolopus. A helicoidal surface pattern is evident. ~ 48, 000.(D) Acid phosphatase in Ceratitis (Diptera) spermatozoa. The activity is evident on the plasma membrane and in the matrix surrounding the tubules. ~120,000.(E) Glycogen on the link-heads in cumpodea (Diplura). Thi&y’s method. ~75,000.(From Baccetti and Dallai, 1972.)

346

B. BACCETTI

Fig. 14

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347

the distal half by a glycoprotein material which undergoes tanning, thus stiffening the entire tail segment (Baccetti et al., 1971b). In a few cases (e.g. in Trichoptera and Drosophila), the tubules are the first to end among the axonemal tubules (Baccetti et al., 1970e; Perotti, 1969). b. The radial links. Besides arms, the doublets also possess other projections which are directed centripetally (Fig. 4). Cross-sectional profiles suggested that they were radial laminae, which was, in fact, the earliest interpretation. They are radial links consisting of straight rays some 325 A long and 70-125 A thick (Behnke and Forer, 1967; Perotti, 1969; Warner, 1970) more or less orthogonally oriented to the fibre and alternatively spaced at 320 A and 560 A . When viewed longitudinally, each closely adhering pair is separated by a wide interval from the other. On the two opposite sides of the flagellum, the radial links are staggered 200 A from each other in a helical coil (Warner, 1970). An exactly comparable situation was reported in Chlamydomonas (Hopkins, 1970), hence it may possibly be of general occurrence.

3. Accessory Tubules These are characteristic, apart from a few exceptions, of the most evolved insects, from Campodeidae Diplura upwards. Their structure and size are closely reminiscent of those of the two central tubules (Fig. 9A, B). However, in some species they are thinner, while in other species (Trichoptera, Lepidoptera) they are thicker (over 300 A in diameter). In cross-section they show 13 globular units in their wall as in the two central tubules (Phillips, 1 9 6 6 ~ )Where . their diameter is wider, these units are in greater numbers; for instance in a Fig. 14. Histochemistry of the tail of insect spermatozoa. (A) Glycogen (arrows) in the central and accessory tubules of Bacillus rossius sperm. ThiCry’s method. ~75,000.(From Bigliardi et Q L , 1970.) (B) Glycogen (arrows) in central and accessory tubule ofhlystacides azurea (Trichoptera) sperm. Thikry’s method. ~90,000.(From Baccetti et al., 1970e.) (C) Pepsin treated sperm tail of Nemoura cinerea (Plecoptera). Doublets and mitochondria1 derivatives (arrows) appear significantly extracted. ~30,000. (From Baccetti et at., 1970e.) (D) Amylase treated sperm tail of Ceratitis capitata (Diptera). Only the accessory tubules (From Bigliardi et al., 1970:) and the two central ones (arrows) appear extracted. ~60,000. (E) ATPase in the sperm tail of Bacillus rossius (Phasmoidea). The reaction (arrows) 1s positive on the coarse fibres, on the arms of the doublets, on the central sheath and on the accessory bodies. ~60,000.(From Bigliardi e t al., 1970.) (F)UTPase in the same material. The reaction appears similar to that of ATPase. ~60,000.(From Bigliardi et al., 1970.) (G) UTPase in the sperm tail of Ceratitis capitata (Diptera). The reaction is positive only in axonema1 (coarse fibres, arms, central sheath) structures. ~60,000.(From Bigliardi et a/., 1970.)

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B. BACCETTI

Coleopteran, as many as 15 or 16 were counted by Shay et al. (1 969). During spermiogenesis, these tubules arise from laminar outgrowths of the B tubules of the doublets (Cameron, 1965). These projections (Fig. 25A, B) first extend outwards in the form of a groove, then bend and once the tubular shape is achieved they separate from the doublets. Later, the material filling them makes its appearance. Therefore, their major chemical component should resemble that of the B tubules, i.e. tubulin B, but no information about this is available. When viewed longitudinally, projections are also discernible which may possibly connect adjacent accessory tubules. The content of the accessory tubules often resembles that of the central ones. In some species which have hollow accessory tubules, the central ones are hollow as well, e.g. in Plecoptera (Baccetti et al., 1970e). In other species (e.g. among the Diptera mentioned earlier) a globular osmiophilic core is found in both categories (Fig. 9). In rare cases, the central tubules are hollow (Fig. lOE), while an osmiophilic core is found in the accessory ones (Neuroptera: Baccetti et al., 1969c). The invasion by microcylinders, typical of Orthoptera (Kaye, 1964, 1970), is more massive in the accessory tubules; up to 15 units can be found in each tubule. A similar event was also reported in Coleoptera (Cameron, 1965; Baccetti, et al., 1972a) where there are fewer, namely, 6-7 units. The most spectacular behaviour is provided by the accessory tubules containing the glycogen-like polysaccharide (Phasmoidea, Trichoptera, Lepidoptera) as reported by Baccetti et al. (1969a, 1970e, 1972b). This polysaccharide appears as a compact mass which fills up the huge tubules previously described as over 300 A in diameter (AndrB, 1961; Yasuzumi and Oura, 1964). In the spermatid stage, during which the accessory tubules are being filled, nine Golgi vesicles (Fig. 25A, D) surround the axoneme, each concentrating around one tubule. Transfer of material, however, could not be established (Baccetti et al., 1972b). In the flagellum of some species, these accessory tubules arise more apically than the other axonemal units (Phillips, 1 9 6 6 ~ ) Since . they are secreted by the B tubules, they are pushed forward after separating from the latter. As a rule, these accessory tubules terminate towards the tail tip before the doublets and the central units. In a few cases, however, such as Trichoptera or Tenebrio (Baccetti et al., 1970e, 1972a), they are the last ones to end.

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In Diplura Campodeidae a 9 + 9 + 2 arrangement is differentiated early in the spermatid. The accessory tubules soon migrate around it and are arranged in two crescents (Fig. 12B, C), one consisting of five and the other of four units aligned against doublets 1, 7 and 8 on one side only of the axoneme. They are separated by the mitochondrion which is juxtaposed to doublet 9 (Baccetti and Dallai, 1972). Similar behaviour is shown in a thysanuran (Machilidae) in which the two crescent-shaped accessory bodies which flank the axoneme displace the accessory tubules into two series, one consisting of four and the other of five elements (Dallai, 1972). The classical 9 + 9 + 2 arrangement is first found in the highest Thysanura, namely, the Lepismatidae (Bawa, 1964; Werner, 1964), and is retained thereafter by all the Pterygota. B. THE CENTRAL SHEATH

This is a structure which surrounds the two central tubules (Fig. 4). In cross-sectional view it shows a circular profile about 700 A in diameter and ranges in thickness from 150 A , in the sector facing doublet 1, t o 90 4 in the contralateral one facing doublets 5 and 6 (Warner, 1970). In longitudinal sections it appears as a helically coiled band, with a pitch of 120 A (Andre, 1961) or of 160 A (Warner, 1970). This pitch, equal to the period of the projections of the central tubules, suggests that the central sheath is somehow anchored to it, even though the latter does not seem to run, as suggested by longitudinal sections, in exact correspondence to the lateral edges of the central tubules. The central sheath is ill-defined from a chemical standpoint because it is an extremely labile structure difficult to isolate or examine by negative staining. How it comes to be connected to the heads of the radial links remains obscure. It certainly contains (Fig. 14E, F, G) ATPase and UTPase (Baccetti et al., 1972b) and is present when, as in Ephemeroptera (Fig. 1OC), the central tubules are completely absent (Baccetti et al., 1969b). C . THE LINK-HEADS

The presence of nine fibrous units between the central tubules and the doublets was demonstrated in the Flagellates by Gibbons and Grimstone (1 960), who called them secondary fibres. Afzelius (1959), in the sea urchin sperm, interpreted them as juxtaposed

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radial links. But their nature remains obscure. Other elements in favour of the existence of longitudinal filamentous connections between the thickenings of the links lying in the same row, have been provided by Andre (1 96 1) in insect spermatozoa, and by Birge and Doolin (1 969) in vertebrate cilia. Almost all other workers have disregarded this problem. More recently, the presence of secondary longitudinal fibres has been disproved both in the flagellate flagellum (Hopkins, 1970) and insect sperms (Perotti, 1969; Bairati and Perotti, 1970; Warner, 1970) as well as in mammalian and amphibian sperms (Fawcett, 1970). All the data available in the literature related to the secondary fibrils in spermatozoa must be ascribed to the link-heads. The link-heads were first described by Andr6 (1961). In cross section, it was shown by Bairati and Baccetti (1965) that they are bipartite; this finding has been consistently confirmed by careful measurements done recently by Warner (1 970): in cross section they measure 200 A by 280 A . This value (Fig. 9A, B) is valid for the Diptera, a Dipluran Cizmpodea, Coleoptera and Orthoptera (Baccetti and Dallai, 1972; Baccetti et al., 1972a). However, a second arrangement exists (Fig. 24A) in Phasmoidea (Baccetti et al., 1972b) and in another Dipluran, Japyx (Baccetti and Dallai, 1972) in which the bipartite structure is less evident and the link-head is smaller in diameter, i.e. about 150 A by 180 A . They are chemically and structurally rather labile, hence poorly understood. Baccetti and Dallai (1 972) showed histochemically the presence of glycogen (Fig. 13E) at the same sites as both the large and small forms of link-heads in Diplura, while such glycogen deposits are not found in all other insects (see Anderson and Personne, 1970). A similar distribution of glycogen was described in some Gastropoda (Anderson and Personne, 1970). D. THE COARSE FIBRES

In the typical insect spermatozoon, an important material exists between the accessory tubules, which is rarely studied because it is difficult to fix and preserve. Baccetti ( 1963) and Baccetti and Bairati (1964) were able to identify four components in what were called “outer fibres” (Andr6, 1961) in Drosophila and Dacus, one of which obviously corresponds to the accessory tubule. Later, Bairati and Baccetti (1965) after identifying the accessory tubules, regarded the interposed material as amorphous, but viewed the presence of the interposed “X granule” as a unit in itself. Generally, all the material

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35 1

lying between the accessory tubules was considered as amorphous by later workers and neglected by them. However, Daems et al. (1 963), as well as Bigliardi et al. (1970), found ATPase and also UTPase localized in it (Fig. 14E, F, G). In Sarcophaga, Warner (1970) has recently identified two coarse fibres lying between the accessory tubules, thus confirming an interpretation similar to that of Baccetti and Bairati (1964). On the basis of histochemical data partly obtained from Dipteran spermatozoa, both coarse fibres (Fig. 14E, F, G) had ATPase and UTPase activity. However, they are certainly not the same in all the insects. On the basis of the literature on this subject it emerges that in Diptera (Fig. 9A) the coarse fibres are generally as Warner (1970) described them and the same is true for Coleoptera (Baccetti et al., 1972a: Fig. 9B) and Phasmoidea (Baccetti et al., 1972b) (Fig. 24A) and even in Thysanura Lepismatidae (Bawa, 1964). In other cases (Fig. lOE), however, they are much attenuated (Neuroptera, Lepidoptera, Plecoptera) and occasionally (Ephemeroptera: Baccetti et ul., 1969b) entirely absent (Fig. 1OC). A peculiar case is found in Diplura Campodeidae in which

Fig. 15. Biflagellate spermatozoa of Rhynchotoidea. (From Baccetti et aZ., 1969d.) (A) The sperm tail ofMenopongallime (Mallophaga).Two axonemes (a) and two mitochondrial (m) derivatives are evident. ~ 6 0 , 0 0 0 .(B) The sperm tail of Chryptothrips latus (Thysanoptera). The axoneme is composed by 18 doublets and four single tubules.

~135.000.

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the accessory tubules are all assembled against doublets 1, 7 and 8 without intervening material. External to doublets 2, 3, 4, 5 and 6, however, there are a series of associated osmiophilic structures which look like coarse fibres and are not separated by tubules. In the case of Bacillus spermatozoa, various nucleoside-phosphate phosphatases (GTPase, ADPase, CTPase) were identified biochemically in the same fraction (Bigliardi et al., 1970). It may be speculated that these enzymes are also concentrated in the same zone together with ATP- and UTPase, thus being involved in flagellar motility . E. THE AXONEMAL MATRIX

This is represented by all the space lying between the structures listed above and separated from the compartment in which the mitochondria1 derivatives are located, by means of membrane residues originating from the Golgi. This space often appears empty in electron micrographs. However, it contains a number of enzymatic activities detectable histochemically or by means of biochemical assays of the supernatant fraction. Histochemically, the most prominent enzyme (Fig. 13D) in this space is acid phosphatase (Bigliardi e t al., 1970; Baccetti et al., 1970b). Lactic dehydrogenase and phosphorylase are also abundant. They may also be detected on the plasma membrane, though only on the inner cytoplasmic membranes (partly plasma membrane, partly Golgi-derived ones) surrounding the axoneme. These enzymes are concerned with carbohydrate metabolism. Biochemical assays carried out on lactic dehydrogenase show that it is essentially present in the supernatant fraction from an Orthopteran, Aiolopus (Baccetti et al., 1970c) while it is strongly associated with membranes in a Phasmoid, Bacillus rossius, where it is quantitatively more important (Baccetti et al., 1972b). The different localization in the two foregoing instances may indicate a different physiological role, bearing in mind that Aiolopus sperm is capable of aerobic metabolism, while that of Bacillus is devoid of mitochondria, hence of cytochrome oxidase. Other enzymes involved in carbohydrate metabolism, i.e. phosphofructokinase, fructose-diphosphate aldolase and glyceraldehyde-3phosphate-dehydrogenase were identified in Bacillus rossius spermatozoa (Baccetti et al., 1972b). So far, their localization is unknown. This enzyme assembly, along with the presence of a polysaccharide in the accessory tubules, points to the existence of a glycolytic

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Fig. 16. Cross sections of several spermatozoa of the Coccid insect Parlatoria aleae: c, chromatin; m, microtubules; n, nucleus. ~70,000. (From Robison, 1970.)

pathway in the axonemal compartment. As a rule, carbohydrate utilization goes t o completion in the reactions catalyzed by mitochondria1 structures. In the absence of the latter (see next Section) it may be deduced that lactic acid is the end metabolite (Baccetti et al., 1972b). AIP--15

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VII. MITOCHONDRIA

As mentioned in the introduction, one of the essential features of the insect sperm is the peculiar evolution of the mitochondria, which are transformed into elongated structures flanking the axoneme. Their organization, however, is not the same in all insects, neither is it simple nor fully understood. A few very primitive insects exist in which the mature sperm has normal mitochondria which are fused into elongated masses. In others, some impressive mitochondrial transformations have occurred, while others lack mitochondria altogether . A. NORMAL MITOCHONDRIA

We may consider as normal mitochondria those with a moderately opaque matrix free of crystalline materials containing abundant well-developed inner cristae with clear-cut cytochrome oxidase activity. This mitochondrial type (Fig. 21 E) is found only among the most primitive insect groups, namely, in all of the Collembola (Dallai, 1967, 1970) and Diplura (Baccetti and Dallai, 1972), in the primitive Machilidae among Thysanura (Dallai, 1972) and in the most primitive Pterygota (Fig. 1OC), the Ephemeroptera (Baccetti et aL, 1969b). In all these cases the mitochondria, though retaining their normal aspect, undergo complex rearrangements during the spermatid phase. At this stage, mitochondria are numerous, small in size and have a tendency to fuse into a few units, undergoing the same rearrangements that were studied in greater detail in the more evolved mitochondria. These transformations lead to three elongated mitochondria in the sperm tail of Collembola, t o two in that of Diplura and Machilidae and to one in the Ephemeroptera sperm tail. These mitochondria retain both normal structure and function throughout the life of the sperm. In some, among the most involuted spermatozoa (Fig. 27) (e.g. those from the higher Termites, Reticulitermes), the mitochondria are also of the conventional model, namely small in size, numerous and provided with cristae (Baccetti eta]., 1972e). B. MITOCHONDRIA TRANSFORMED INTO DERIVATIVES WITH A CRYSTALLINE CORE

This, the best known mitochondrial category, was first studied by light microscopy (Bowen, 1920). The fine structural details have

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been fully described by Andre (1959, 1962) in Lepidoptera, by Pratt (1968) in a Hemipteran as well as by occasional reports from several other authors. All this data was coordinated by Phillips (1970b) who provided a scheme which can be applied to almost all insects. During the last spermatogonial generations and spermatocyte I prophase, the small spherical mitochondria increase considerably in number, until they fuse into long chains, oriented by the poles, forming a “palisade around the spindle” (Andre, 1962). Thus, meiosis leads to an exact distribution of mitochondria between the two daughter cells, until, during the second telophase, the mitochondria pack together into a skein (Retzius’ Nebenkern, 1904). Then there begins in each spermatid, the slow and conspicuous metamorphosis (Fig. 17) of the mitochondria into the Nebenkern through stages which And& called “twinned”, “chromophobic envelope”, “onion” and “loaf”. During these rearrangements two extremely long, labyrinthically interwoven identical chondrioconts are formed (Pratt, 1968) which eventually separate and come to lie on either side of the incipient axoneme. Together with the latter, the mitochondria develop within a narrow evagination of the plasma membrane against which the nucleus is first located. A cytoplasmic space gradually (Figs 18, 19, and 20) forms between the nucleus and the periaxonemal membrane as the latter is dragged distalwards away from the nucleus by the ring centriole (Fawcett and Phillips, 1970). The two Nebenkern derivatives insert themselves within this space (Phillips, 1970b), progressively elongating and flanking the axoneme. They taper until they become equal in size to the axoneme itself. In the Pterygota there are usually two mitochondrial derivatives (Figs4, 9, and 10) which either remain alike or one of them overtakes the other in length or in width (see Phillips (1970b) for a detailed case history). Occasionally, one of them progressively diminishes until it vanishes, thus the mature sperm possesses but one mitochondrial derivative (Fig. 10A, B). This occurs in the Mecoptera (Baccetti et aZ., 1969c) and Trichoptera (Baccetti et aZ., 1970e; Phillips, 1970b). Likewise in Thysanoptera (Baccetti et al., 1969d) (Fig. 15B) and in Diptera (e.g. Psychodidae, Baccetti et al., 1972c) (Fig. 26) only one mitochondrial derivative was found. In these cases, however, the spermatid mitochondria fuse directly into a single mass rather than two initial ones. When the two mitochondrial derivatives have flanked the axoneme for almost its entire length (instances of their equalling the axoneme in length are extremely rare) (Folliot, 1970), two important changes

Fig. 1 7

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take place. First of all (Fig. 22C), the cristae become aligned, regularly spaced at right-angles to one side of the major axis of the derivative, looking like a series of short lamellae. Then within the mitochondrial matrix some masses of material contained in regular granules, with a paracrystalline structure (Fig. 21 D), make their appearance. These were studied first by Andr6 (1 962). Subsequently they were resolved by Meyer ( 1964) and by Phillips (1 966a) using negative stains. This paracrystalline material invades the space left free of cristae in all the insects so far studied. It consists (Fig. 14C) of a protein (Baccetti et al., 1969c, 1970e) whose three-dimensional organization was suspected of varying according t o species (Meyer, 1964). It contained various types of longitudinal and transverse periodicities (the most common being 450 a) and different unit arrangements, which generally are hexagonally spaced. Two diverse crystalline organizations within the same mitochondrion have been found. The example quoted by Phillips (1970b) is not relevant, however, since the mitochondria described ‘are in reality an ordered extramitochondrial material (Mazzini, 1970). Intramitochondrial crystals probably have a pattern which is very common to many, if not all, insect orders. In Fig. 22(A, B), the same structure is demonstrated in a Dipteran and in a Coleopteran sperm. This material does not arise spontaneously within the mitochondria (Baccetti et ul., 1972a). During the last phases of spermiogenesis it was noticed by Baccetti and his collaborators (1 972a) that the Golgi cisternae come into contact with the mitochondrial wall and deposition of the early proteinaceous aggregates takes place at this site (Fig. 21A, B, C). This material is of unknown significance. In several species, repeated histochemical investigations (Bigliardi et ul., 1970; Baccetti et al., 1970b, 1972a) have shown that cytochrome oxidase is confined to the short cristae (Fig. 22C) and that the pro teinaceous matrix contains none of the enzymes concerned with respiration, glycolysis or the hydrolysis of triphosphate nucleosides. In all the spermatozoa provided with mitochondria, therefore, there are two pathways for energy production in the flagellum. Fig. 17. Various stages of mitochondrial condensation and Nebenkern formation in the spermatid of Murgantia hisrrionicu (Rhynchota). (From Pratt, 1970.) (A) and (B). Early cluster or “twinned” Nebenkern. ~18,000.(C) Later cluster or “twinned” Nebenkern. ~16,000.(D) “Small sheet” or “chromophobic envelope” Nebenkern. x 17,000. (E) “Four layered” or “onion” Nebenkern. x 16,000. (F) “Two layered” or “loaf” Nebenkern. x 17,000.

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Fig. 18. Young spermatid of Tenebrio (Coleoptera). The ring centriole (rc) is still very close to the centriole (c) whereas the Nebenkern (n) are far away (cf., Fig. 19). ~30,000. (From Baccetti et aL, 1972a.)

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Fig. 19. Later spermatid of Euchistus (Rhynchota). The ring centriole (rc) is some distance from the centriole (c) and the two Nebenkerns (nb) are nearer than in Fig. 18. ~ 1 3 , 0 0 0(From . Phillips, 1970b.)

Normal respiration in the mitochondria1 compartment is by means of the Krebs cycle and anaerobic glycolysis which occurs in the axonemal compartment at the expense of the polysaccharide contained within the accessory tubules.

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Fig. 20. A still later stage spermatid of Euchistus (Rhynchota). The Nebenkern (ntd surrounds the axoneme between the ring centriole (rc) and centriole (c) ~13,000. (From Phillips, 1970b.) C. ABSENCE OF MITOCHONDRIA

Only one insect group provided with a typical flagellum is devoid of mitochondria. It is the order Phasmoidea (Fig. 24A), in which the genera Bacillus (Baccetti et al., 1972b) and Clitumnus have been studied. The young spermatid has a moderate complement of

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36 1

Fig. 21. Mitochondria in insect sperms. In (A), (B) and (C) a system of narrow membranes (m) originating from Golgi vesicles (g) penetrates between the axoneme (a) and mitochondrial derivatives (md) of Tenebrio spermatozoon, fusing with its limiting membranes (arrows). The inner mitochondrial crystallization (c) occurs near the region of first contact with the Golgi membranes. ~90,000.(From Baccetti et al., 1972a.) (D) Frozen-etched preparation of Tenebrio spermatozoon. The crystalline material filling the mitochondrial derivatives is evident (arrows). ~60,000. (From Baccetti et aL, 1972a.) (E) A normal mitochondrion (m) in a mature insect sperm (Campodea). ~48,000.(From Baccetti and Dallai, 1972.)

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Fig. 22. Crystallized mitochondrial derivatives in insect sperms. (A) Crystallized material of Drosophila (Diptera) after phosphotungstic acid negative staining. ~260,000.(From Bairati and Perotti, 1970.) (B) Crystallized material of Tenebrio (Coleoptera) after uranyl acetate negative staining. ~306,000.(From Baccetti et aL, 1972a.) (C) CytochromeC oxidase in the peripheral region (containing cristae) of Tenebrio mitochondrial derivatives (From Baccetti etaZ., 1972a.) (arrows). The crystalliie region (c) is negative. ~60,000.

mitochondria (which show no tendency to evolve into a Nebenkern), which possess inner cristae rich in cytochrome oxidase (Fig. 24B). At this stage, cell respiration is conducted normally. Subsequently, mitochondria undergo degeneration and are definitely absent in the spermatozoon. Biochemical assays showed that both cytochrome oxidase and succinic dehydrogenase activities were extremely low (Bigliardi et al., 1970) and were probably the result of contamination by spermatids. Oxygen consumption in sperm preparations, as

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determined in the presence of cytochrome c and ascorbate, is also absent (Baccetti e t al., 1972b). As reported in the preceding section, metabolism in the axonemal compartment is definitely reduced to glycolysis taking place under strictly anaerobic conditions. Both mitochondria or any form of mitochondrial derivative are absent from the category of non-flagellate sperms (Termites of the genus Kalotermes and coccids). In both cases (Figs 27 and 28), the spermatozoon is supplied with a rich microtubule complement (see below). In the sperm of coccids, which are the best studied, ATPase is present (Moses, 1966). Therefore, an active energy source, though different from oxidative phosphorylation, may be postulated (Ross and Robinson, 1969). In all these cases, the solution of the energetic problem may be analogous to that found in Phasmoidea spermatozoa. However, this is still an open question.

VIII. ACCESSORY ORDERED FLAGELLAR BODIES

One of the essential features of the sperm tail in insects is the fact that, besides the mitochondrial derivatives, the axoneme is flanked by one or more elongated accessory structures of paracrystalline texture. These structures are reminiscent of the compact protein core which often invades the mitochondrial matrix in Pterygotes. However, some do not seem to be related to them, either chemically or functionally. In addition, several categories may be identified. A. STRUCTURED BODIES FLANKING NORMAL MITOCHONDRIA

This condition occurs only in the most primitive insects, in which mitochondria do not undergo their characteristic metamorphosis. In some Thysanura (Dallai, 1972) two long crystalline rodlets were described (Fig. 23B), which flank the flagellum throughout its length. They have a crescent shape when viewed in cross section. In longitudinal sections they display a paracrystalline texture with a periodicity of 200 A . In Ephemeroptera two elongated crystalline bodies (Fig. lOC) are also seen to flank the single mitochondrion. They have an average periodicity of 160 A . They seem to arise in close contact with the Golgi cisternae (Baccetti et aZ., 1969b). Their function and chemical composition is unknown.

3 64

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Fig. 23. Patterns of accessory flagellar bodies in insect sperms. (A) Tenebrio molitor (Coleoptera). ATPase present around the two bodies (b) and absent from mitochondrial derivatives (m) ~120,000.(From Baccetti et al., 1972a.) (B) Muchilis distincta (Thysmura). The accessory bodies (b) are much larger than mitochondria (m). ~ 6 0 , 0 0 0(From . DdS, 1972.) (C) Cixius netvows (Rhynchota). The accessory bodies (b) are as developed as the mitochondrial derivatives (m) ~35,000.(From Folliot and Maillet, 1970.) (D) Megouru viciue (Rhynchota). Spermatid: The accessory bodies (b) originate in contact with Golg membranes (g). ~ 6 0 , 0 0 0(From . Mazzini, 1970.)

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€3. STRUCTURED BODIES FLANKING THE MITOCHONDRIAL

DERIVATIVES WITH A CRYSTALLINE MATRIX

I. Connected with the Centriole Adjunct

These formations occur in greater number and are better known than the preceding ones. In the spermatid, they make their appearance first as caudal expansions of the centriole adjunct. As they become progressively enlarged and surrounded by microtubules, they flank the axoneme and mitochondrial derivatives and end up embedded in the flagellum. A single one is found in Plecoptera (Baccetti et al., 1970e) and in Cicindela (Werner, 1965). Two symmetrical ones are present in Tenebrio (Baccetti et al., 1972a) and in Aphaniptera (Baccetti, 1968). They are primarily of a protein nature, with a carbohydrate core, demonstrated histochemically in Tenebrio. Again in Tenebrio, these structured bodies (Fig. 23A) are seen to be rich in ATPase and UTPase at least in a cortical halo consisting of globular units of 80 A . They show a compact organization in their central zone where the polysaccharide is located. The function of these formations still has to be assessed; however, the occurrence of ATPase seems to suggest some role in flagellar motility. Their origin is also unknown. The Golgi-derived cisternae never come into contact with them (Baccetti et al., 1972a). In Cicindela, Werner (1965) has documented the origin of the centriolar adjunct and its derivative from a caudal zone of the nucleus, where the nuclear membrane is highly porous. 2. Products of the Golgi Complex This section deals with the origin of the two accessory formations found in Homoptera , which are symmetrically positioned, like the mitochondrial derivatives, on either side of the axoneme. They often exhibit bizarre profiles (Fig. 23C) and a crystalline texture. They were studied in Delphacids by Herold and Munz (1967), in Cicadellidae by Phillips ( 1970b), in several Auchenorhyncha by Folliot and Maillet (1970) and in Aphids by Mazzini (1970). Their chemical characteristics and function are obscure. Only in Aphids has their origin from the Golgi-derived cisternae (Fig. 23D) been ascertained (Mazzini, 1970). C. STRUCTURED BODIES REPLACING MITOCHONDRIA

This is an extreme event typical of Phasmoidea (Bigliardi et al., 1970; Baccetti et al., 1972b). In this order, mitochondria disappear

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Fig. 24(A) The sperm tail of Bacillus rossius: ab, accessory bodies; at, accessory tubules; cf, coarse fibres; ct, central tubules; d, doublets. Mitochondria are lacking. x 120,000, (From Baccetti e t al., 1972b.) (B) Cytochrome-C-oxidasein a normal mitochondrion (m)of the spermatid of Bacillus. Seligman et al. 's method. ~ 6 0 , 0 0 0(From . Baccetti et al., 1972b.)

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during spermiogenesis and are replaced by two large bodies (Fig. 24A). These consist of alignments of proteinaceous material arranged in periodically juxtaposed small laminae and longitudinal filainents. In all sections, along almost the entire length, they appear J-shaped. They arise from the Golgi at the spermatid stage (Fig. 25B, C), are made up of proteins and are extremely rich in ATPase and UTPase. Their role is important as regards the type of locomotion, which in this case is peculiar as it follows almost circular trajectories. Although they do not appear to be structured in alternative fashions according to their thickness and do not change organization when the spermatozoon is naturally or experimentally relaxed, it was supposed that they function in contraction in view of their enzymatic equipment (Baccetti et al., 1972b). IX. SPERMATOZOA POSSESSING A DOUBLE FLAGELLAR APPARATUS OR BEING DEVOID OF IT

In relation to the rule of 9 which seems. to dominate doublet arrangement, mention has already been made of the huge flagellum of Sciara coprophila studied by Phillips (1966a) and by Makielski (1966). This has 70-90 doublets (Fig. l l ) , each flanked by an accessory tubule, which are derived from a large centriole spirally coiled around a single mitochondria1 derivative. This case seems to depend on the particular type of spermatogenesis (Metz, 1938) which leads ultimately to a single spermatid with a chromosome complement, two Xs and one or two heterochromatic chromosomes, corresponding t o the germinal tissue. In other sperm categories, described earlier, either the absence or an excess number of both central and accessory tubules can be found. A different case is represented by germ cells which either have two normal flagellar devices or where the flagellum is completely absent. A. THE PAIRED SPERMATOZOA

This is a peculiar situation found occasionally in the animal kingdom. It is generally known in Thysanwa Lepismatidae (Bawa, 1964; Werner, 1964) as well as in the coleopteran Dytiscus marginalis (Ballowitz, 1895), the gastropod Turritella terebru (Retzius, 1906; Idelman, 1960) and in American marsupials (Biggers and Creed, 1962; Phillips, 1970c, d). In Thysanura Lepismatidae, membrane fusion unites sperms two by two at the level of the nucleus.

Fig. 25

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Movement is possible only under this condition and isolated sperms are non-motile. In these insects, unlike Dytiscus, the sperm pair remains united in the female genital tract. What happens when the sperm penetrates the egg is obscure (Bawa, 1964). The type of fusion of the membranes at the nuclear level also requires further investigations. Phillips ( 1 970c, d) has observed in Opossum that at the moment of sperm pairing a gap junction arises between the two sperm membranes. B. SPERMATOZOA POSSESSING TWO AXONEMES

This is a characteristic feature of the whole group of orders known as Rhynchotoids (Fig. 15A, B); namely, the Psocoptera, Mallophaga, Anoplura, Thysanoptera and Rhynchota. In all instances, the doubling of this structure concerns only the axonemes and the centriole adjuncts, with the exception of. the mitochondria1 derivatives which are two in number (almost in .all cases) or even a single one (in Thysanoptera). Psocoptera generally have a single axoneme (Phillips, 1969); however, in one species, Atropos puZsatorium (Baccetti et al., 1969d), a high percentage of biflagellate sperms are found among the normal ones. It should be kept in mind, with regard to this, that among insects the accidental appearance of sperms with two axonemes is relatively frequent. This may be experimentally enhanced by mercaptoethanol treatment (Friedlander and Wahrman, 1965). It was reported by Baccetti et al. (1 969d) that, among Mallophaga, two equal axonemes consistently originate from two closely apposed centrioles lying beneath a rather enlarged nucleus. An analogous condition is found in Anoplura, among which Pediculus (Ito, 1966) and Haemutopinus (Baccetti et al., 1970a) have been studied. Among Thysanoptera (Fig. 15B) two axonemes are still encountered (Baccetti et al., 1969d); here, however, the accessory tubules, radial links and central sheath are wholly absent and the four central tubules, together with the 18 doublets, haphazardly form into a single bundle. It is curious that in such an arrangement Fig. 25. The origin of accessory tubules and accessory bodies in the spermatozoon of Bacillus rossius. (From Baccetti ef nl., 1972b.) (A) The tubules B of the doublets form a long outer arm, (a), and are surrounded by a system of Golgi (g) cisternae symmetrically disposed. ~ 6 0 , 0 0 0 .(B) The outer arms of the B tubules produce the accessory tubules (arrows). A Colgi (g) membrane is folded forming the precursor of an accessory body (b). ~180,000. (C) A later spermatid, with complete accessory tubules (at) and the rudiment of an accessory body (b) arising from a Colgi vesicle (g). ~90,000.(D)Migration of Cold vesicles (g) towards the axoneme. ~ 6 0 , 0 0 0 .

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the axoneme still retains its motility (unpublished observations on Thrips). Among Rhynchota the condition with two axonemes has the tendency to disappear. The heteropteran, Pyrrhocoris (Furieri, 1963b) has two axonemes which lie side by side, one longer than the other-a situation not found in other species. Other Heteroptera, e.g. Gelastocoris (Payne, 1966) and Nepa (Werner, 1966) have a single axoneme. The same is true of the most evolved Rhynchotoids, i.e. the Hornoptera, whose highest forms, the coccids, have non-flagellate spermatozoa. The presence of two axonemes, therefore, does not seem to be a haphazard event; it is a condition which distinguishes the primitive Rhynchotoid orders but disappears in the vast majority of the species in the most evolved orders. The type of motility acquired is rather peculiar and disorderly (see below). It is difficult to foresee what its selective advantage can be. C. NON-FIaAGELLATE SPERMATOZOA

This seemingly paradoxical condition appears, as far as I know, only in four insect groups (in one of which in two different forms). In three cases it appears to be associated with non-motility (the sperm being passively displaced), in the other two cases there are alternative organelles concerned with movement. Among the latter, the most widely known example is that of coccids mentioned above. In these Rhynchotes the syncytial condition which is shared by all insects during a given period of spermiogenesis (64 spermatids in Drosophila; 256 in Dacus-according to Baccetti and Bairati (1964); 128 in Atropos-according to Baccetti (1967)-and in Trychopteraaccording to Phillips (1970b); always increasing by the 2nd power) is retained by the adult sperms. In the light microscope Nur (1962) observed sperm bundles consisting of 16 elements in Pseudococcus obscurus, 32 in Eriococcus and Parlatoria, 64 in Puto. Moses and Coleman (1964) found in Steatococcus tuberculatus 32 spermatozoa in every bundle, each of them exhibiting a set of parallel microtubules surrounding the nucleus two and a half times. Robison (1966, 1970) and Ross and Robison (1969) have studied Parlatoria oleae, Pseudococcus obscurus, Matsucoccus bisetosus, Stomacoccus plantani, Kermes sp. and Put0 albicans. In the last species, spermatozoa generally exhibit a “cork-screw” shape. A motile apparatus, consisting of an alignment of 20-250 microtubules, spirals around a central chromatin axis (Fig. 16). Nuclear membrane,

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acrosome, mitochondria, centrioles and axoneme are all absent. The microtubules contain ATPase (Moses, 1966) and their arrangement always follows asymmetrical patterns. According to Robison (1 970) these features suggest that the microtubule bundle is associated with sperm motility. Ross (1 97 1) points out that the cork-screw shape is attained by virtue of a second microtubule system helically coiled around the spermatid, thereby moulding its characteristic outline. On the contrary, in one case in the Termites, replacement devices do not exist, hence the absence of a flagellum is accompanied by non-motility. In another instance replacement structures are available. The loss of flagella is progressively acquired in the course of evolution of the different families, the most primitive of which seem to possess normal flagellate spermatozoa. Some examples were succinctly described at the light microscopical level by Springhetti (1963). In the more primitive Mastotermitidae and Termopsidae, the spermatozoon is elongated and bears a flagellum; it is likely to be of the orthopteroid type encountered also in Embioptera (Baccetti et ul., 1972e). But in Kalotermitidae and Rhinotermitidae, spermatozoa appear to lack the flagellum. Baccetti et al. (1972e) have described this spermatozoon in a specimen from the first family, Kalotermes flavicollis and in one belonging to the second family, Reticulitermes Zucifugus. In the latter (Fig. 27) the spermatozoon is a severely involuted cell. It has a compact almost perfectly spherical shape. The acrosome is absent. At the posterior pole, a small cytoplasmic rim accommodates a few mitochondria of a conventional pattern, namely spheroidal in shape and small in size. Also present are two extremely short parallel cylinders looking like centrioles, but which are actually two truncated axoneme apices lacking the two central tubules and only provided with nine doublets at their periphery. These two flagellar anlagen do not protrude from the roundish sperm body which is made up solely of a head and unable to move. In Kulotermes (Fig. 28) a much more bizzarre shape is encountered. During spermiogenesis, the nucleus of each spermatid is condensed and flattened into an ovoid shape by a set of microtubules which surround it. The plasma membrane is separated from the nuclear membrane by a most tenuous interspace in which the microtubules are seen to run in a single array even in the mature sperm. The plasma membrane is displaced at four angles into four long arms which are also completely occupied by microtubules. Thus, apart from the acrosome, this sperm is devoid of flagellar anlagen and mitochondria. This situation closely resembles that

Fig. 26. Cross sections of mature sperms of Thelmatoscopus albipunctatus: a, acrosome; m, mitochondria1derivative; n, nucleus. The axoneme is lacking. ~120,000.(From Baccetti e t al., 1972c.)

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Fig. 21. The spermatozoon ofReticulitermes lucifugus.~ 6 0 , 0 0 0 .

Fig. 28. The spermatozoon of Kalotermes jlavicollis. ~ 3 9 , 5 0 0 .

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found in coccids. How this spermatozoon moves, if it does, is unknown. However, the long flattened arms crammed with microtubules suggest a pattern of undulating membranes. In the Diptera Psychodidae, Baccetti et al. ( 1 9 7 2 ~have ) examined several species, in which a common behaviour was pointed out. Their spermatozoon (Fig. 26) looks like a rather stiff long needle, with a multilayered cortical zone. This is actually the result of an association of the plasma membrane with the secretory products occurring in the testis lumen, which surround each germ cell. Inside this theca there is a nucleus which is highly elongated, compact and wholly or partly enveloped by a profuse material which is derived from the Golgi and may be viewed as an acrosome. Embedded in the acrosome is a small mitochondria1 derivative which has few cristae. These spermatozoa, which distinguish the whole Psychodidae family, are also completely immotile. In the genus of Protura, Ementornon, the spermatozoon has the form of a disk when contained in the testes. The circular nucleus is perispherical and flattened mitochondria lie in the central area. After ejaculation, the cell assumes the form of a cup. X. MOTILITY A. MOTILE MECHANISMS

The problem of insect sperm motility is centred around an understanding of ciliary motion of which it is a variation. Indeed, there are a series of variations of increasing complexity which parallel an increase in structural complexity of both supporting and contractile tail structures. Most attempts to explain ciliary or flagellar motion have so far focused on the flagellum of flagellate Protozoa or on the sperm tail of the sea urchin, both of which have many similarities. They both have a plain axoneme of the 9 + 2 pattern. Some experiments have been performed on the bull spermatozoon, whose axoneme is more complex ( 9 + 9 + 2) but whose general pattern agrees with the preceding model, being a sperm with a large head and a short tail. From the overall information thus far collected (Holwill, 1966; Brokaw et al., 1970; Brokaw, 1971) two conclusions are evident: (1) In Protozoa and in sea urchin sperm, motion consists of consecutive waves propagated along the tail thus resulting in a planar beat. Bull spermatozoa, however, show a helicoidal motion.

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(2) In spermatozoa these waves originate from a point situated in the centriole zone (Bradfield, 1955). This was demonstrated by amputations performed by Bishop and Hoffmann-Berling (1959), Goldstein and Brokaw (1968) and Goldstein (1 969) with a laser microbeam, and by Van Herpen and Rikmenspoel's (1969) indirect estimates concerning the size of this control centre on the basis of its susceptibility to X-rays and protons. In Flagellata, on the contrary, the amputated portion of the flagellum can beat and a different origin of waves must be supposed (Goldstein et al., 1970). At present, any explanation as to the role played by the single fibrillar components in motility and the mechanisms involved in wave triggering and propagation, are a matter of speculation. The presence of directly contractile structures has been debated. Gray (1 955) and Machin (1 958) considered that they were not associated with any precise flagellar component, whereas others have identified them with the two central tubules (InouC, 1959) or with the nine doublets which are the only universally occurring structures also having ATPase in their arms (Bradfield, 1955; Silvester and Holwill, 1965). Satir (1961) advanced a hypothesis based on the twocomponent mechanism of muscle contraction. He suggested that a myosin-like component might exist in the tubules and an actin-like one in the matrix, which for the first time is thus believed to be involved in flagellar motility. Along this line, again under the influence of muscular contraction, Satir (1968) and Brokaw (1968, 1971) put forward a new view on a sliding model, excluding the matrix and proposing (Satir) a sliding of each B subfibre with respect to the others. This fact cannot be demonstrated in sperm tails, where the orientation of the bent tip is difficult to determine. A separation between A and B subfibres has occasionally been described at the tip of the tail in Insect spermatozoa, but this does not demonstrate any form of sliding. Another hypothesis has been put forward by Schreiner ( 197 1) on purely mathematical grounds. He excludes contraction along any type of tubule and postulates the existence of welding points between the matrix and motile tubules along the flagellum, which would lead to the displacement of the straight segments during wave propagation. The active part of the movement would therefore be the recovery stroke. Nothing is known about this in the insect sperm flagellum where the recent demonstration of actin filaments, apparently independent of the microtubules (Behnke et al., 1971), suggests the presence of a muscle-like

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Fig. 29. Sequences of frames from high-speed cinefilms showing flagellar beat of different insect sperms. (A) Ctenocephulus mnis: 9 + 2 model. Total time: 0.21 s. x380. (B) Gryllotulpu gryllotulpu: 9 + 9 + 2 model with a rigid caudal tip (t). Total time: 0.43 s. x560. (C) Huematopinus suis: 9 + 9 + 2, the axoneme twinned in each sperm. Total time: 0.21 s. x790.

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mechanism. In connection with the helical shape of the waves, the structures which exhibit a helical arrangement around the sperm main axis may be thought to play an important role in the progression of the contractile events. These structures are the central sheath and the radial links. It is interesting to recall that the doublets run parallel to the sperm main axis. Motion pictures of sperms from different species show at least three forms of movement which seem to correspond to a structural hierarchy. The spermatozoa (9 + 2 axonemal pattern) of some species show a simple, almost planar wave of low frequency with a wave length as long as, or longer than, that of the flagellum. Tails are generally inextensible and a single wave type arising in the centriolar zone is propagated along them. The example examined by us is the spermatozoon of Aphaniptera (Fig. 29A). This has the simplest type of motility which closely resembles that of flagellates, marine sperms and cilia in general. At a second level of complexity there is a helical wave (owing t o two harmonic movements of equal amplitude). This model (Fig. 29B) is typical of all the species possessing the 9 + 9 + 2 formula, with accessory tubules and ATPase-rich accessory fibres. This type of movement and structure occurs in many insect and mammalian sperms. At the third level of complexity there is a combination of the two types of undulation. This is the form of movement which occurs in spermatozoa having a 9 + 9 + 2 tail plus accessory bodies endowed with ATPase activity. In some cases these bodies enable the sperm to course a doubly helical trajectory which remains straight (Tenebrio: Fig. 30A), in other cases their shape compels them to follow always doubly helicated trajectories in a helical course (Bacillus: Fig. 30B). A detailed study on sperm motility (Baccetti et al., 1972b) deals with this last form of movement which incorporates the preceding ones. The relevant deductions are as follows. Initiation of motility takes place in the centriolar region and has been identified in the axoneme apex which is supplied with laminae showing ATPase activity. This motor unit is called initiation motor (it must be remembered that the mature insect sperms lack a centriole). In this region it is postulated that consecutive small range revolving movements occur around the central axis, leading to related displacements between the bundle of tubule apices and the centriole adjunct. This torsional impulse propagates passively along the tail, through the assembly of tubules by virtue of their inextensibility. The internal viscosity of this system involves some energy expendi-

Fig. 30

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twe during the transmission of the movement. For this reason the first deformation stops almost at once and is of limited mechanical extension. This deformation, however, is enough to trigger a contraction in all the axonemal structures possessing ATPase activity (central sheath, arms, coarse fibres), possibly reacting with the actin units and thus propagating the contractile wave along the flagellum; this wave propagation is an active one. Tubules function only to propagate the mechanical deformation to consecutive levels. This wave is not reflected, but it dies away near the end of the flagellum. Upon it, at a following moment, a second wave of greater amplitude is superimposed. It arises when the oscillation frequency of the initiation motor nears the natural oscillation frequency of the structures which surround the axoneme. The resulting resonance enhances the oscillation amplitude of the outer structures until a mechanical deformation is also produced outside the axoneme, such as to evoke the active reactions of the extraaxanemal bodies which also are endowed with ATPase activity. The greater structural consistency of these organelles results in wave propagation towards the caudal end, as if it was an elastic wave which is reflected by the tail tip. This reflection occurs in a reverse direction, since it may be tentatively postulated that the surrounding medium acts as a boundary for the tail. The helicoidal trajectory is determined by the non-rectilinear shape and non-linear reactivity of the structure of the sperm tail. When the above observations are extended to more simpler cases, it may be deduced that where extraaxonemal bodies are not available a single cylindrical-helicoidal wave type arises (simple 9 + 9 + 2 insects). When the ATPase-containing coarse fibres lying between the accessory tubules are also absent, the quasi-planar wave type is evoked. This wave form has a much lower frequency, but a greater length being as long as the sperm tail and it is characteristic of the 9 + 2 model (first type of movement, as for instance among Aphaniptera). Other motility patterns, related to a particular flagellar architecture have emerged. As already mentioned, Gryllotalpa (Baccetti et al., 1971b) has a tail divided into two segments. The first has a Fig. 30. Sequences of frames from high-speed cinefilms of flagellar beat of different insect sperms. (A) Tenebrio molitor: 9 + 9 + 2 model with little accessory bodies: the trajectory is double helicated, but linear. h, Head. Total time: 0.17 s. x460. (B) BuciZlus rossius: 9 + 9 + 2 model with enormousaccessory bodies. The trajectory is double helicated and circular. h, Head. Total time: 0.09 s. x430.

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conventional 9 + 9 + 2 pattern, without accessory tubules and moves according to the general insect model. The second, with sclerified tubules, is completely rigid (Fig. 29B). The biflagellate Rhynchotoid sperm also moves in an irregular fashion. For instance, in Anoplura a rather ill-coordinated movement is found (Fig. 29C), although it belongs to the category of a single cylindrical helicoidal wave, short and frequent (general type), as suggested by the absence of accessory bodies. Movement of the complex syncytium of sperms in Coccids is most peculiar (Robison, 1970) and can possibly be regarded as helicoidal, like the motion of flagella (on which, however, the various wave types have not been studied). Since the accessory bodies are lacking, it should be ascribed to the two simpler categories, possibly to the second one. B. METABOLIC ASPECTS OF MOTION

In the preceding sections it was seen that in insect spermatozoa an important energy source is due to intense anaerobic glycolysis occurring in the axonemal complex. Normal respiration effected in the mitochondria1 compartment, when present, may be added to it. In all cases the stored polysaccharide is glycogen, which usually accumulates in the accessory tubules at the spermatid stage. The mature spermatozoon can thus call upon an endogenous store of carbohydrate. However, since a high carbohydrate level exists in both the seminal fluids and the spermatheca secretions, these may be thought of as being somehow a part of the material metabolized by the spermatozoon, despite the fact that the uptake of carbohydrate, or even indications of any type of pinocytosis, have never been demonstrated. Indeed, in many cases, sperm locomotion was observed even without carbohydrate administration (Hughes and Davey, 1969). In normal respiring sperms, oxygen is an element which regulates their motion. It may be experimentally demonstrated not only that these sperms are immotile under anaerobic conditions, but also that they migrate towards oxygen gradients. It was suggested that the tracheae-rich female conceptacula can thereby direct the movement of spermatozoa (Rao and Davis, 1960). Spermatozoa devoid of mitochondria are insensitive to oxygen (Baccetti e t al., 1972b). Hydrogen concentration also exerts an important effect on sperm motility: an optimum between pH 7.0 and 7.4 was demonstrated in Cimex by Rao and Davis (1 969). Richards (1963a) was the first to show that in Periplaneta sperm

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motility is a function of temperature and attains a maximum at 39". Davey (1958) has made similar observations. In Bacillus sperm (Baccetti et al., 1972b) motility was enhanced up to 50"C, but then it decreased though still remaining high up to 60°C. At this elevated temperature, however, few sperms were seen to survive. Temperature does not seem to appreciably influence wave length, but it does enhance both wave frequency and velocity. Motility is obviously affected by the osmotic pressure of the medium. In Periplaneta, Davey (1 958) reported a maximum activity with a tonicity exceeding 400 mosmoles. Later, lower tonicities were tested (Hughes and Davey, 1969). Generally, in flagella, altered conditions of viscosity affect the frequency, but not the form of beat (Brokaw, 1963, 1966; Holwill, 1965). Nevertheless, the high viscosity seems to reduce to two dimensions the normal threedimensional motion of bull spermatozoa (Rothschild, 1961). Studies on this problem in insect sperms are in progress. . C. THE PROBLEM OF SPERM CAPACITATION

This subject has been little investigated in insects. As observed by Hughes and Davey (1969) the general concept we can reach at present is that in some species fully mature sperms are transmitted to the female, while in other species their maturation is achieved in the female organism, thereby leading to a form of capacitation. In Periplanetu, for example, the extraacrosomal space is reduced in the spermatheca (Hughes and Davey, 1969). Likewise, in Sciuru a portion of the Nebenkern is eliminated (Makielski, 1966). In coccid insects the spermatozoa originally assembled under syncytial conditions become free individuals (Robbins, 1965). As stated earlier (Section 11), the most outstanding transformations occurring in the spermatheca concern the digestion and remoulding of the glycoprotein materials overlying the plasma membrane (Payne, 1934, in a hemipteran; Riemann, 1970; Riemann and Thorson, 1971; Phillips, 1971, in many Lepidoptera; Renieri and Vegni, 1972, in Iocusts). A form of capacitation may perhaps be recognized in these aspects, which in the moths Trichoplusiu and Anugustu culminates in the formation of an extracellular rod-like crystalline structure running parallel to the sperm, embedded in two thick crystallized sheaths. All this complex comes into being exclusively within the female genital tract and it originates from the material which formed the laminated appendages and the satellite body (Riemann, 1970; Riemann and

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Thorson, 197 1). The fine structure of the penetrating spermatozoon still requires more detailed study. XI. SPERMATOZOA POLYMORPHISM AND GENETICS

After the earliest work by von Siebold (1836), who discovered an atypical spermatogenesis associated with a typical one in a Mollusc, a number of investigations have been focused on the typical two-fold spermatogenesis found in many invertebrates; in particular in Lepidoptera where it was brought to light by Meves (1903). Fain-Maurel ( 1966) and Zylberberg ( 1969) have summarized its most relevant aspects. Among the various insect orders, when an atypical sperm line exists, it is generally a diploid or polyploid hyperpyrene line (Richards, 1963b; Bouix, 1963) because of variations in the ratio of DNA to nuclear proteins (Ansley, 1958). On the contrary, in Lepidoptera either an apyrene line is found when the nuclei are entirely eliminated during spermiogenesis, or an oligopyrene line when some nuclear residues persist (Zylberberg, 1969). Hyperpyrene spermatozoa have a normal length, but much thicker heads and tails as compared to normal sperms. They have been little studied as yet; the apyrene ones in Lepidoptera have been the object of recent research on the parts of Zylberberg and of Phillips (197 1). In these latter cells the nucleus does not undergo its typical condensation during the spermatid stage, as is true of eupyrene sperms; it remains spherical, glides caudal along the flagellum and then is eliminated. The acrosome does not form and the centriole comes to lie in the anterior cell portion. The corpuscle, interpreted by Zylberberg (1969) as a centriole, is conversely interpreted as a cap of undefined material by Phillips (1971). Both workers have found defective mitochondria1 derivatives in the apyrene line. In addition, Phillips (1 97 1) finds hollow instead of solid accessory tubules and, as already stressed, an absence of lacinate appendages around the plasma membrane. The presence or absence of the nucleus is not therefore related to the absence of any other organelle (excluding the acrosome, which forms on the nucleus itself), but rather is seen to affect normal organization in the spermatocyte stage. In fact, the cytoplasm has already received all its necessary information from the nucleus, and the spermatid nucleus is dormant (Olivieri and Olivieri, 1965). Many important papers on this aspect have appeared describing the long series of investigations on Drosophila mutants lacking the Y chromosome (Meyer, 1964, 1968; Kiefer, 1966;

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Anderson, 1967). Drosophila heydei does not progress beyond the spermatocyte I stage (Hess and Meyer, 1963); in Drosophila melanogaster, on the contrary, spermiogenesis begins without going to completion. All the spermatozoa of these X-0 males possess: ( 1) ill-organized mitochondria1 derivatives, sometimes supernumerary ones, with an anomalous crystalline core; (2) distorted or broken axonemes, which may lack some of its units; (3) bundles each having an overall smaller number of sperms which have shorter tails because they are immature. On the other hand, in the XYY males, spermatozoa are twice as long as normal ones (Meyer, 1968). Hence, in Drosophila. the Y chromosome seems to be endowed with a given number of fertility factors essential for the development of normal functioning spermatozoa. These factors must come into play during spermatocyte I stage since in normal X males spermatozoa are also normal. In this phase, similar loops to those of the lampbrush chromosomes are seen to form. In special mutants in which the Y chromosome is shorter (Bairati and Baccetti, 1966) and lacks one or more loops, sperms do not undergo maturation, thus resembling those from X-0 males. These individuals are sterile (Hess and Meyer, 1968). A particular mutant with a short Y (KL- 1-), studied by Kiefer (1969), has appgrently normal and motile spermatozoa and is capable of fertilizing the female. However, these spermatozoa degenerate within the female. They probably bear biochemical deficiencies which cannot be detected at the morphological level. In summary, the hypothesis is forwarded that the Y chromosome does not code for the proteins required in the formation of the sperm organelles, but governs the coordination of the various synthetic and morphogenetic steps (Meyer, 1970). Similar malformations have been induced by heat (Anderson, 1967) and X-rays (Hess, 1965). Altering the haemolymph composition by adding K + and Mg2+ (Meyer, 1969) also induces similar effects. All these altered features may in fact be viewed as phenocopies of Y deficiencies. What happens in the normally X-0 species (Orthoptera, Protenor) under the same experimental conditions has not been studied. Neither is it known whether a dimorphism between X-sperms and 0-sperms is detectable in those species in which a large X occurs, while their autosomes are minute. Almost all the genetics of spermatozoa still need studying.

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ACKNOWLEDGEMENTS

The original research reported in this article was supported by C.N.R. I am indebted to Drs. A. Bairati, jr., R. Dallai, R. Folliot, P. L. Maillet, M. Mazzini, E. Perotti, D. M. Phillips, S . Pratt and W. G. Robison for permission to use figures. REFERENCES Afzelius, B. (1959). Electron microscopy of the sperm tail. Results obtained with a new fixative. J. biophys. biochem. Cytol. 5,269-278. Allen, R. D. (1 968). A reinvestigation of cross-sections of cilia. J. Cell Biol. 37, 825-83 1. Anderson, W. A. (1 967). Cytodifferentiation of spermatozoa in Drosophila melanogaster. The effect of elevated temperature on spermiogenesis. Molec gen. Genet. 99, 257-273. Anderson, W. A. and AndrB, J . (1968). The extraction of some cell components with pronase and pepsin from thin sections of tissue embedded in an epon-araldite mixture. J. Microsc. 7,343-354. Anderson, W. A. and Ellis, R. A. (1967). Cytodifferentiation of the crayfish spermatozoon: acrosome formation, transformation of mitochondria and development of rnicrotubules. 2.Zellforsch. mikrosk. Anat. 77,80-94. Anderson, W. A. and Personne, E. (1 970). The localization of glycogen in the spermatozoa of various invertebrate and vertebrate species. J. Cell Biol. 44, 29-5 1. AndrB, J. (1 959). Etude au microscope electronique de l’evolution du chondriome pendant le spermatogknkse du Papillon du Chou Pieris brassicae. Annls Sci. nat. Zool. 12 Ser., 283-308. AndrC. J. (1961). Sur quelques ddtails nouvellement connus de l’ultrastructure des organites vibratiles. J . Ultrastruct. Res. 5 , 86-1 08. AndrB, J. (1962). Contribution 2 la connaissance du chondriome: Btude de ses modifications ultrastructurales pendant la spermatogBnkse. J. Ultrastruct. Res. Suppl. 3, 185 pp. AndrC, J. (1963). Some aspects of specialization in sperm. In “General Physiology of Cell Specialization” (G. Mazia and A. Tyler, eds), pp. 9 1-1 15. McGraw-Hill, New York. Andrt, J. (1965). A propos d’une leCon sur la Limule. Annls Sci. Clermon t-Ferrand 26, 2 7-3 8. Andrb, J. and Bernhard, W. (1 964). The centriole and the centriolar region. In “XIth International Congress Cell Biology”. Providence, R.I.9. AndrB, J. and ThiBry, J.-P. (1963). Mise en Bvidence d’une sous-structure fibrillaire dans les filaments axonkmatiques des flagelles. J. Microsc. 2, 7 1-80. Ansley, H. R. (1958). Histones of mitosis and meiosis in Loxa flavicollis (Hemiptera). J. biophys. biochem. Cytol., 4, 59-62. Austin, C. R. (1948). Function of hyaluronidase in fertilization. Nature, Lond. 162. 63-64.

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