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Spermiogenesis of Anthonomus grandis Boheman, the cotton boll weevil, was studied by transmission electron microscopy. The basic morphologic features investigated and illustrated were: acrosome, nucleus, fibrous masses, microtubules, and mitochondrial derivatives. A centriole adjunct was observed to surround the basal body (centriole derivative) early in spermiogenesis, but it subsequently disappeared. In the sperm tail, one mitochondrial derivative developed into a rod-like structure that inserted into a concavity at the base of the nucleus and terminated near the end of the tail. This mitochondrial derivative may provide a rigid base for the undulating membrane. In addition, the attachment of the rod to the nucleus may serve in a manner analogous to the function that has been suggested for the centriole adjunct: it may anchor the head to the tail. A possible abnormal condition may arise when the mitochondrial derivative rod fails to insert into the sperm nucleus.

Spermatogenesis in the honey bee (Apis mellifera L.) differs from the process in diploid insects in several ways. The primary spermatocytes in the haploid drone undergo an abortive first meiotic division and, further, an unequal division of the secondary spermatocyte occurs. The result is two spermatids rather than the typical four. Associated with the first meiotic division, a centriole replication process occurs resulting in supernumary centrioles; the latter are eliminated from the cells through cytoplasmic blebs prior to the second meiotic division. During the centriole replication process, several single annulate lamellae appear to be differentiated from pre-existing lamellae of endoplasmic reticulum. An amorphous granular material is observed to surround the replicating centrioles, and it is similar to material located in the pores of the annulate lamellae and nuclear envelope. Because of the specific orientation which the annulate lamellae illustrate relative to the replicating centrioles, it is postulated that they are somehow involved in the accumulation of material utilized in centriole replication. The secondary spermatocyte spindle has associated with it a nuclear “wall” composed of alternate vesicular and compact layers of endoplasmic reticulum. The innermost layer of this wall appears to give rise to the spermatid nuclear envelope. Spermatocytes are interconnected by intercellular bridges throughout the process of spermatogenesis and it is postulated that this syncytium synchronizes the division process. Spermiogenesis in the honey bee is similar to that observed in a number of other insects.

In this paper the spermatozoon of a plecopteran and of a trichopteran is described. The former is an “orthopteroid type” spermatozoon, with the following peculiarities: acrosome lying in lateral position with respect to the nucleus; proteinaceous rodlet arising from the adjunct centriole and flanking the two mitochondrial derivatives in the tail; accessory fibers of the axial-filament complex hollow even at maturation. This spermatozoon is therefore quite different from Odonata and Ephemeroptera sperms previously described, and it represents the simplest case of structure of the outer accessory fibers known in insects. The spermatozoon of the trichopteran exhibits the following characteristics: absence of an acrosome, a third supernumerary proteinaceous slender fibril between the two principal central ones, only one mitochondrial derivative entirely occupied by parallel transverse cristae and lacking a crystalline axis. The outer accessory fibers of the flagellum are 300 A in diameter and, in this respect, closely recall those of Lepidoptera, comparatively reexamined in this paper. Like the principal central units, these fibers are filled with glycogen-like polysaccharides and proteins, and they are longer than the 9 principal peripheral doublets. This model of Trichoptera and Lepidoptera outer fibers is quite different from the pattern hitherto described in other insects.

Epoxy embedding methods of Glauert and Kushida have been modified so as to yield rapid, reproducible, and convenient embedding methods for electron microscopy. The sections are robust and tissue damage is less than with methacrylate embedding.

The mature spermatozoon of Spadella cephaloptera belongs to the filiform type. At the front end it contains a membraneous cap, which may be an acrosomal structure, and some beaded sacs. A crystal structure is found in these sacs in spermatid stages. Near this front end there is a centriolar structure. A very elongated mitochondrial derivative runs through most of the sperm. In the central half of the sperm there is a structure composed of some longitudinal sacs lying alongside the axonema. The nucleus is situated in the hind end, just in front of a free flagellum. The spermatozoon is highly unique, but to some extent it agrees with spermatozoa found among arthropodes and gastropodes.

Resume Les transformations du chondriome pendant la spermatogenese ont ete etudiees a l'aide du microscope electronique chez une cinquantaine d'especes animales zoologiquement tres variees. Elles sont decrites en detail pour trois d'entre elles: Rat, Testacella (Mollusca, Gastropoda), Pieris (Insecta, Lepidoptera). Chez ces especes le chondriome, d'ultrastructure habituelle au depart (spermatogonies), se multiplie abondamment et subit des modifications qui leguent au spermatozoide des corps mitochondriaux d'ultrastructure inhabituelle. Les faits les plus saillants de cette evolution sont: I. Chez le Rat: (1) l'apparition d'une substance nouvelle, claire, nommee pseudomatrice, dilatant les espaces intermembranaires des cretes (espaces intracristaux); (2) l'augmentation d'opacite des espaces matriciels, avec une reduction concomitante de leur volume qui amene une inversion des contrastes par rapport a l'ultrastructure normale; (3) l'expulsion de la pseudomatrice; (4) l'allongement et l'enroulement autour du flagelle de ces mitochondries modifiees, qui finalement s'alignent les unes derriere les autres, tout en conservant leur individualite, en deux helices, jointives et de pas constant, entre lesquelles s'intercalent localement une troisieme, et parfois une quatrieme helices; II. Chez la Testacelle: (1) l'apparition d'une pseudomatrice, accompagnee d'une reduction et d'une opacification des espaces matriciels, comme chez le Rat; (2) la disposition en spheres emboitees des lames matricielles; (3) le groupement periflagellaire de ces mitochondries a structure concentrique; (4) leur fusion en un seul chondriosome, de dimensions gigantesques; (5) l'evanouissement des structures de l'internum mitochondrial, veritable metamorphose au cours de laquelle membranes, matrice, pseudomatrice et granules disparaissent, tandis qu'apparaissent des domaines ordonnes; (6) la croissance des domaines ordonnes, qui s'organisent finalement en deux manchons concentriques dont la structure, paracristalline, a ete analysee, et dont la nature, non etablie avec certitude, est supposee proteique; il est suggere que les elements constitutifs du reseau paracristallin, petits bâtonnets creux de 90 A. representent un etat particulier des proteines respiratoires; (7) la demixtion de substances, supposees lipidiques en majorite, d'ultrastructure variee, qui forment trois helices distinctes (dont deux sont jumelles) logees entre les deux manchons paracristallins; cites sont errones, douteux ou insuffisamment prouves. (4) Cette augmentation du chondriome est favorable a la recherche des modalites de la multiplication mitochondriale; les constatations faites sur le Rat conduisent a emettre l'hypothese que de nouvelles mitochondries se forment par induction au voisinage immediat d'une mitochondrie preexistante, ou par croissance et scission d'une mitochondrie-mere; elles forment alors des groupements particuliers de mitochondries qui terminent leur maturation, puis se dispersent dans le cytoplasme. Dans les deux cas, la presence d'une mitochondrie initiale serait necessaire. III. Cytochimie et biochimie. Les colorations classiques apres fixation, la coloration supravitale au vert Janus B et la detection de l'activite succino-deshydrogenasique a l'aide du tetrazolium nitro-bleu, mises en parallele avec les images de la microscopie electronique, concordent a montrer que, a la fin de la spermatogenese, les enzymes respiratoires membranaires sont non seulement presentes, mais concentrees, meme dans les cas ou la morphologie est profondement aberrante. Les implications physiologiques de cette concentration enzymatique sont discutees.

The mitochondrial derivative of the sperm cell of the Gastropod Helix is endowed with a compartment loaded with glycogen (Personne et André, 1964). The aim of this work is to establish whether this mitochondrial glycogen is synthesized in the mitochondrial derivative itself or elsewhere in the cell. For this purpose, living sperm were first incubated in a medium containing glucose 1-phosphate, then fixed, sectioned, and stained specifically for polysaccharides by the phosphotungstic acid technique or the periodic acid-thiosemicarbazide-silver proteinate technique. Comparison with controls shows that a synthesis of glycogen occurred during the time of incubation. It can be inferred from this result that an amylophosphorylase system controlling the metabolism of polysaccharides is present in the mitochondrial derivative itself. Results obtained with the iodine technique indicate that the original glycogen molecules are elongated during the experiment. It seems probable that the amylophosphorylase system demonstrated here accounts for at least a part of the proteinaceous coat morphologically detectable around each individual glycogen particle.

Although 9-membered centrioles are found in somatic tissues of Sciara, the centriole which lies at the spindle pole of the second meiotic division in male Sciara is composed of a row of approximately 70 short tubules in an oval array. Shortly after telophase of this unequal division, in the daughter cell destined to undergo spermiogenesis, microtubules become confluent with the tubules of the centriole. These tubules have the same density as other cytoplasmic microtubules after glutaraldehyde-OsO4 fixation and, like them, are not preserved with OsO4 fixation. As the centriole, now with approximately 70 attached, posteriorly directed, doublet tubules, migrates from the polar to the apolar end of the nucleus to take a final position in an oval groove which forms in the nuclear envelope, the tubules lengthen and become demonstrable after OsO4 fixation and more electron opaque than other cytoplasmic microtubules following glutaraldehyde-OsO4 fixation. Later, a singlet tubule appears peripherally to each doublet of the oval and 4 "arms" develop at specific sites on the tubules. Posteriorly, where the oval of tubules becomes discontinuous and forms a spiral, the arrangement of arms is different and the singlet tubules are lacking. Dense solid bodies develop inside this odd flagellum and become enclosed by a smooth double membrane. A single mitochondrial derivative has three components: a central area of homogeneous, moderately electron-opaque, proteinaceous material; a peripheral ring of cristae; and a crystalloid which is specifically oriented with respect to the flagellar tubules.

Though the fagellum of Sciara sperm arises from a blepharoplast and is characterized by doublet tubules with arms, it differs markedly from the familiar type of flagella in the number and arrangement of its tubules. The axial filament complex in sperm from the testis of Sciara consists of approximately 70 doublet tubules, each with an associated singlet tubule. Near the nucleus these tubules are displaced in an oval array. Posteriorly the oval breaks and coils from one free end so that the axial filament complex at posterior levels has the form of a spiral. The singlet tubules do not extend the full length of the sperm but terminate in order from inside the spiral. Farther posteriorly the axial filament complex reverses the direction of coiling, and the doublets terminate from outside the spiral. Four arms are specifically positioned on the singlet and doublet tubules. A single mitochondrial derivative extends most of the length of the sperm; it consists of a large mass of proteinacious material, a crystalloid located adjacent to the axial filament complex, and peripheral cristae. In the female genital tract, sperm undergo gross morphological changes which include sloughing of practically all the mitochondrial material except the crystalloid, repositioning of the crystalloid, and uncoiling and subsequent recoiling of the axial filament complex into a different configuration. From analysis of serial sections it was determined that the orientation of arms, when the axial filament is viewed from base to tip, is the same as in conventional flagella.

The mitochondrial derivative of the sperm of the gastropod pulmonate Biomphalaria glabrata was studies to ascertain succinic dehydrogenase localization cytochemically. Two techniques were compared. One technique depends on a tetrazolium salt that yields an osmiophilic formazan upon reduction. The other technique is dependent on the reduction of copper ferricyanide. The effects of several electron transport inhibitors were studied. The reaction product observed in the matrix of the mitochondrial derivative using the former technique is sensitive to rotenone and is believed to be nicotinamide adenine dinucleotide-dependent. The reaction product observed in the intracristal spaces using the copper ferricyanide method is insensitive to rotenone and is believed to cytochemically demonstrate succinic dehydrogenase in this material.

1. The study of certain structures, like the acrosome and Nebenkern, in the developing sperms of Arvelius is facilitated by the fact that they are present in greatly enlarged dimensions in certain testicular lobes while retaining normal size in adjoining lobes. 2. The dictyosomes or Golgi bodies in the spermatocytes are stained red by the PAS treatment. They are assembled to form the similarly stained acroblast in the spermatid, and the acrosome that is finally evolved is also PAS-positive in its color reaction. All the evidence indicates that in the process of acrosome formation there is a direct utilization of dictyosomal material which is already present two cell generations earlier. 3. The substance responsible for the red staining of the dictyosomes and acrosome is a polysaccharide with a 1,2 glycol grouping. There is also direct evidence for the presence of small quantities of proteins which probably form a complex with the polysaccharides. This complex is similar or perhaps identical with a mucopolysaccharide. 4. The Nebenkern, which is a mitochondrial derivative, does not contain polysaccharides with a 1,2 glycol grouping. On the other hand, it contains considerable amounts of proteins which, in contrast to those of the acrosome, are easily digested by pepsin. The evidence does not support the suggestion, frequently made, that the acrosome and Nebenkern are closely interrelated. 5. The enzymatic nature of the acrosome and its possible role in fertilization and sterility are discussed.

The axial filament of Sciara coprophila does not conform to the usual 9 + 2 filament pattern but consists, rather, of as many as 76 pairs of filaments which decrease in number from the anterior to the posterior region of the sperm. It is first seen at the base of the head in the shape of an indented oval. The axial filament varies in configuration along the remaining length of the sperm as one whorl or two connected whorls of filament pairs. The other structures of the sperm revealed by the light and electron microscopes are a homogeneous, dense, spear-shaped nucleus, a row of spherical dense bodies in the middle piece enclosed by the axial filament and of unknown nature and function and a single mitochondrial derivative. The mitochondrial nebenkern derivative consists of a large electron transparent region bordered by cristae and a smaller paracrystalline region located adjacent to the axial filament. The derivative arises as paracrystalline material in a medial nuclear indentation. The electron transparent material is first seen at the anterior end of the middle piece. Unlike other known insect sperm, but reminiscent of sperm capacitation in mammals, sperm maturation is completed in the spermathecae of Sciara 7 to 9 hours after insemination. It consists of the acquisition of sperm motility and elimination of the electron transparent region of the mitochondrial nebenkern derivative. The electron microscope reveals in mature sperm that the axial filament doublets have changed configuration and consist of a single whorl which encloses the paracrystalline rod. The process by which the major portion of the nebenkern derivative is eliminated occurs in four identifiable stages. Since sperm maturation does not appear to be intrinsically controlled, factors in the spermathecal fluid may play a role in its completion.

Publisher Summary 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 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.

The ultrastructure of the paracrystalline mitochondrial derivative of the Drosophila spermatozoon has been investigated in sperms stored for different intervals after mating within the genital apparatus of inseminated females and in sperms which penetrated the egg. The paracrystalline lattice remains structurally intact even after the most prolonged storage. Only after fertilization does the derivative exhibit structural modifications which following a characteristic pattern lead to the formation of lamellar bodies present up to the onset of the cellular blastoderm stage of the egg development. The behaviour of the mitochondrial derivative during the sperm storage, its fate at fertilization, and the absence of cytochrome c oxidase activity from its crystalline texture, as demonstrated by cytochemical tests, suggest that the typical biochemical activity of mitochondria is not a major function of this highly differentiated sperm component. The functional significance of the derivative is discussed in relation to a possible role at fertilization and to sperm metabolism.