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Spermatogenesis is a dynamic process of cellular differentiation that generates the mature spermatozoa required for reproduction. Errors that arise during this process can lead to sterility due to low sperm counts and malformed or immotile sperm. While is estimated that 1 out of 7 couples encounter infertility, the underlying cause of male infertility can only be identified in 50% of cases. Here, we describe and examine the genetic requirements for missing minor mitochondria (mmm), sterile affecting ciliogenesis (sac), and testes of unusual size (tous), three previously uncharacterized genes that are predicted to be components of the flagellar axoneme. Using Drosophila, we demonstrate that these genes are essential for male fertility and that loss of mmm, sac, or tous results in complete immotility of the sperm flagellum. Cytological examination uncovered additional roles for sac and tous during cytokinesis and transmission electron microscopy of developing spermatids in mmm, sac, and tous mutant animals revealed defects associated with mitochondria and the accessory microtubules required for the proper elongation of the mitochondria and flagella during ciliogenesis. This study highlights the complex interactions of cilia-related proteins within the cell body and advances our understanding of male infertility by uncovering novel mitochondrial defects during spermatogenesis.

The Drosophila melanogaster pAbp gene encodes the cytoplasmic poly(A)-binding protein (PABP). Cytoplasmic PABPs participate in the metabolic pathways of the mRNA: translation initiation and termination, cytoplasmic polyadenylation/deadenylation, mRNA stability, mRNA degradation. Despite the extensive biochemical and structural characterization, relatively little is known about the biological roles of PABPs in the processes of cellular development and differentiation. In Drosophila spermatogenesis, posttranscriptional mechanisms of gene regulation play an important role, so cytoplasmic PABP can have significant function in this process. Deletion of PABP leads to embryonic lethality. However, some flies carrying combinations of mutant pAbp alleles survive but display male sterility and show defects in spermatogenesis. It has previously been shown that hypomorphic pAbp mutations cause a number of meiotic defects, abnormalities of Nebenkern formation. These data provide an insight into the effect of pAbp mutation on the individual events of spermatogenesis, but they do not cover the entire process. We studied spermatogenesis in pAbp allele heterozygotes by transmission electron and fluorescent light microscopy. We showed that cellular mitochondria were the primary structural target of the mutation. Abnormal mitochondria were less structured, swollen, had transparent matrix and depleted cristae. Further mutation had a polymorphous effect and induced anomalies in the ultrastructure of mature spermatocytes, defects in Nebenkern formation and division, axial complex formation, shutdown of spermatogenesis during spermatid elongation. Thus our data show a significant role of PABP in structural transformations of male germ cells during entire spermato-genesis.

The red palm weevil (RPW), Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae), is a pest that is rapidly spreading across the globe. Here, the ultrastructure of R. ferrugineus spermatogenesis and sperm are described. The histology of the testis, sperm ultrastructure, and spermiogenesis were investigated using light and transmission electron microscopy. The differentiation of spermatids was observed to occur within spermiogenetic cysts. Inside each cyst, the spermatids were at the same stage of maturation. During early stages, mitochondria aggregated, fused, and elongated beside the growing flagellar axoneme, while the proacrosome transformed into a triple-layered acrosome, with a perforatorium, acrosomal vesicle, and extra-acrosomal layer. The centriolar adjunct was present in early spermatids but was absent from later spermatid stages and sperm. The sperm's tail displayed a typical axoneme with a 9 + 9 + 2 microtubule arrangement, 2 mitochondrial derivatives of unequal size, and 2 acc...

Background In Drosophila early post-meiotic spermatids, mitochondria undergo dramatic shaping into the Nebenkern, a spherical body with complex internal structure that contains two interwrapped giant mitochondrial derivatives. The purpose of this study was to elucidate genetic and molecular mechanisms underlying the shaping of this structure. Results The knotted onions (knon) gene encodes an unconventionally large testis-specific paralog of ATP synthase subunit d and is required for internal structure of the Nebenkern as well as its subsequent disassembly and elongation. Knon localizes to spermatid mitochondria and, when exogenously expressed in flight muscle, alters the ratio of ATP synthase complex dimers to monomers. By RNAi knockdown we uncovered mitochondrial shaping roles for other testis-expressed ATP synthase subunits. Conclusions We demonstrate the first known instance of a tissue-specific ATP synthase subunit affecting tissue-specific mitochondrial morphogenesis. Since ATP synthase dimerization is known to affect the degree of inner mitochondrial membrane curvature in other systems, the effect of Knon and other testis-specific paralogs of ATP synthase subunits may be to mediate differential membrane curvature within the Nebenkern.

In Drosophila, the ubiquitin ligase Hyd (hyperplastic disc) is required for regulation of cell proliferation during development [Martin et al. (1977) Dev Biol 55, 213-232; Mansfield et al. (1994) Dev Biol 165, 507-526]. Earlier, we demonstrated that the Drosophila tumour suppressor Merlin participates not only in imaginal discs proliferation control, but also performs a separate Nebenkern structural function in Drosophila spermatogenesis [Dorogova et al. (2008) BMC Cell Biol 9, 1. Here, we show that the hyd mutants also have spermatogenesis defects: chromosome condensation and attachment to the spindle, centrosome behaviour and cytokinesis in meiosis. The process of spermatid elongation was also greatly affected: nuclei were scattered along the cyst and had an abnormal shape, Nebenkern-axoneme angular relation and attachment was distorted, axonemes themselves lost correct structure. Since Hyd and pAbp protein families share a common PABC [poly(A)-binding protein C-terminal] protein domain, we also studied spermatogenesis in pAbp homozygotes and found defects in cytokinesis and spermatid elongation. However, our study of hyd and pAbp genetic interaction revealed only the phenotype of defective nuclei shape at the final stage of spermatid differentiation. So, the PABC domain is unlikely to be responsible for meiotic defects. Thus, our data document that, in addition to the tumour suppressor Merlin, another tumour suppressor, Hyd, also has a function in spermatogenesis.

Mitochondria undergo dramatic rearrangement during Drosophila spermatogenesis. In wild type testes, the many small mitochondria present in pre-meiotic spermatocytes later aggregate, fuse, and interwrap in post-meiotic haploid spermatids to form the spherical Nebenkern, whose two giant mitochondrial compartments later unfurl and elongate beside the growing flagellar axoneme. Drp1 encodes a dynamin-related protein whose homologs in many organisms mediate mitochondrial fission and whose Drosophila homolog is known to govern mitochondrial morphology in neurons. The milton gene encodes an adaptor protein that links mitochondria with kinesin and that is required for mitochondrial transport in Drosophila neurons. To determine the roles of Drp1 and Milton in spermatogenesis, we used the FLP-FRT mitotic recombination system to generate spermatocytes homozygous for mutations in either gene in an otherwise heterozygous background. We found that absence of Drp1 leads to abnormal clustering of mitochondria in mature primary spermatocytes and aberrant unfurling of the mitochondrial derivatives in early Drp1 spermatids undergoing axonemal elongation. In milton spermatocytes, mitochondria are distributed normally; however, after meiosis, the Nebenkern is not strongly anchored to the nucleus, and the mitochondrial derivatives do not elongate properly. Our work defines specific functions for Drp1 and Milton in the anchoring, unfurling, and elongation of mitochondria during sperm formation.

Mitofusins comprise a family of evolutionarily conserved, nuclear encoded mitochondrial guanosine triphoshatases that control mitochondrial fusion and morphology. The fuzzy onions (fzo) and Drosophila mitofusin (dmfn) genes, which encode the only Mitofusin homologs in Drosophila are differentially expressed during development. Dmfn-mRNA was widely expressed during embryogenesis accumulating in the mesoderm and endoderm during gut development, during oogenesis with transcripts maternally deposited into the early embryo and in the male germ line, where dmfn-mRNA was expressed in spermatogonia, spermatocytes and early spermatids. In contrast, expression of the fzo was tightly restricted to the male germ line, with mRNA accumulation in spermatocytes and early spermatids. In addition, expression of dmfn and fzo in the same cell type, primary spermatocytes, was under control of different regulatory mechanisms.

Mitochondria are essential organelles of developing spermatids in Drosophila, which undergo dramatic changes in size and shape after meiotic division, where mitochondria localized in the cytoplasm, migrate near the nucleus, aggregate, fuse and create the Nebenkern. During spermatid elongation the two similar mitochondrial derivatives of the Nebenkern start to elongate parallel to the axoneme. One of the elongated mitochondrial derivatives starts to lose volume and becomes the minor mitochondrial derivative, while the other one accumulates paracrystalline and becomes the major mitochondrial derivative. Proteins and intracellular environment that are responsible for cyst elongation and paracrystalline formation in the major mitochondrial derivative need to be identified. In this work we investigate the function of the testis specific big bubble 8 (bb8) gene during spermatogenesis. We show that a Minos element insertion in bb8 gene, a predicted glutamate dehydrogenase, causes recessive male sterility. We demonstrate bb8 mRNA enrichment in spermatids and the mitochondrial localisation of Bb8 protein during spermatogenesis. We report that megamitochondria develop in the homozygous mutant testes, in elongating spermatids. Ultrastructural analysis of the cross section of elongated spermatids shows enlarged mitochondria and the production of paracrystalline in both major and minor mitochondrial derivatives. Our results suggest that the Bb8 protein and presumably glutamate metabolism has a crucial role in the normal development and establishment of the identity of the mitochondrial derivatives during spermatid elongation.

The ultrastructure of spermatogenesis and spermatozoa was studied in Timema poppensis Vickery & Sandoval, 1999, a putative basal taxon of Phasmatodea. The apical portion of testis follicles consists of spermatogonial cells with polymorphic nuclei. Primary spermatocytes display very short primary cilia originating from the peripheral centrosomes. Early spermatids develop a conspicuous “nebenkern” consisting of fused mitochondria. They have a single peripheral centriole with microtubular triplets, which expresses a 3.6-μm-long cilium featuring a 9 + 2 axonemal pattern. In a later stage, the centriole and the ciliary shaft displace toward the inner part of the cytoplasm by an infolding of the plasma membrane. Mature spermatids exhibit a derived centriole with microtubule doublets devoid of dynein arms, which is equipped with a dense arc-like outer structure. Ciliary degeneration was not observed during spermiogenesis. Spermatozoa are short flagellate cells about 55–60 μm in length. They are characterized by a three-layered acrosomal complex. The distinctive bell-shaped morphology of the acrosome vesicle is likely an autapomorphic trait of Timema. The flagellum has a 9 + 9 + 2 axoneme, two accessory bodies, two flattened cisterns, and two elongated mitochondrial derivatives. Results support the hypothesis that Phasmatodea, comprising Timema + Euphasmatodea, form a monophyletic group. The presence of 17 protofilaments in the wall of accessory microtubules and the flattened configuration of the flagellum are potential apomorphic groundplan features of the order. Within Phasmatodea, a key evolutionary divergence was from the conventional insect spermiogenesis and sperm structure of Timema, to the unusual spermiogenetic process and peculiar sperm structure of Euphasmatodea. As a result, Timema retains more sperm character states found in the polyneopteran ground pattern, while Euphasmatodea have evolved outstanding sperm autapomorphies, like the loss of mitochondria and flattened cisterns, and the presence of strongly expanded accessory bodies.

Spermatogenesis in Drosophila mojavensis (Diptera : Drosophilidae) begins with the proliferation of a primary gonial cell, leading to the formation of a cyst with 16 primary spermatocytes that subsequently enter a growth phase towards the end of meiosis. As their development proceeds, primary spermatocytes assume a characteristic species-specific appearance regarding the morphology of their nucleus. Meiosis produces a syncytium of 64 spermatids, each one of which bears one nucleus and one compact mass (nebenkern), composed of mitochondria. During spermiogenesis, the newly formed spermatids undergo a structural transformation that leads to the formation of the mature sperm: the spherical nucleus is reduced to a 0.1 × 75 μm cylinder and the nebenkern separates into 2 mitochondrial derivatives, which elongate to 2400 μm, while associated with the axoneme. Both mitochondrial derivatives of the mature sperm, although disparate in size, are relatively large and have paracrystalline bodies that occupy a great part of their mass.

Summary The ultrastructure of the spermatogenic cycle of the ground dwelling carabid beetle, Pasimachus subsulcatus and the spermatids of the sympatric P. strenuus were examined using standard transmission electron microscopy. It is hypothesized that spermiogenesis in P. subsulcatus is at least in part facilitated by microtubule-assisted elongation of nebenkern derivatives and nuclei, as indicated by peripheral microtubules surrounding these structures in the spermatids. Mature spermatozoa of P. subsulcatus and spermatids of both species are characterized by two symmetrical elongate mitochondria, extending from the nucleus to nearly the full length of the fiagellum. Mitochondrial cristae are arranged perpendicularly to the long axis of the cell in evenly spaced parallel folds. The flagellum of both species is characterized by the typical 9+2 arrangement of microtubules with an additional peripheral singlet of nine microtubules. The morphology of P. subsulcatus sperm is similar to that of other insects des...

The aberrant sex-ratio mutation in D. simulans used for this study is a temperature-sensitive autosomal recessive. Homozygous males raised at 16 degrees C produce about 2% of F1, but those raised at 26 degrees C, have a normal sex ratio. Ultrastructural studies of spermatogenesis have revealed many anomalies in the germ cells of flies raised at 16 degrees C, but the same flies raised at 26 degrees C had few anomalies. The earliest spermatogenic stage with noticeable abnormalities was the primary spermatocyte. In later stages there were pronounced abnormalities in nucleoli, chromatin condensation, nuclear shape, Golgi complex, acrosome, and microtubules. There is asynchronous differentiation of spermatids within a bundle. Some of the abnormalities encountered are disorganization or loss of microtubules of the axoneme, degenerating nebenkern derivatives, and increased numbers of lysosomes, multilamellate bodies, and multivesicular bodies. At the lower temperature, more than half of the sperm within the same bundle were found in different stages of degeneration. Genetic analysis suggests that the sex-ratio gene causes abnormalities and degeneration of most of the Y-bearing sperm. However, counts of abnormal sperm at the ultrastructural level indicate that some X-bearing sperm must also undergo degeneration. These observations show that the sex-ratio gene is of variable penetrance at different temperatures in the primary spermatocyte and of differential penetrance in X- and Y-bearing sperm.

We have examined the ultrastructure of post meiotic eupyrene sperm mat­ uration in testes of prepupae, pupae, and adult Heliothis virescens (F.) males. Emphasis was placed on the structures present in the elongating sperm tail. In each young spermatid, each cell has a nucleus, acrosome derivative, and numerous mitochondria in a large volume of cytoplasm. These mitochondria coalesce to form a body, the nebenkern. As the sperm cell begins to elongate, the nebenkern divides into two mitochondrial derivatives (MDs) of unequal size. The MDs have an outer and inner membrane, and as they elongate and coil in the sperm tail, cristae develop on the inner membrane. Concurrent with cell elongation is the disappearance of most of the cytoplasm present in the cell. The axial filament has a typical insect system of 9 + 9 + 2 tubules and is present along most of the length of the sperm tail as are the MDs. A prominent system of tubules, the manchette, forms around the MDs in a later stage of development. Two extracellular structures, the satellite body and the radial mantle, also undergo a complex series of changes during the maturation of the sperm cell.

In a study of spermiogenesis of Locusta migratoria , the differentiation process has been divided into ten developmental stages. A clearly recognizable character is chosen as the main feature to define each stage, so that in the forthcoming investigations involving experimental treatment, the chosen feature may serve as a guide line. All cell organelles are described in each of the stages. During the first three stages, the mitochondrial nebenkern is formed. It divides into two derivatives while the proacrosome transforms into a conical-shaped structure. From stages 5 to 9 the chromatin rearrangment leads to a homogeneous compact and slender nucleus, while the residual cytoplasm is sloughed off and while the axoneme reaches its definitive configuration. The spermatodesms, formed during stage 9, are transferred into the seminal vesicles during stage 10. Then all microtubules, other than the axonemal ones, disappear. The extracellular fibrous coat of the sperm cell is formed in an early stage. It differentiates very progressively and reaches its high degree of organization only during the last stage.

The effects of spermatogenesis of a series of continguous non-overlapping Y-chromosome deficiencies were examined using both the light and electron microscope. The deficiencies were constructed by combining elements of different X-Y translocations; they subdivide the Y into seven segments, six of which are required for male fertility (four in the long arm and two in the short arm). Spermatogenesis was examined from the primary spermatocyte through to the formation of mature sperm and the earliest departures from normal development identified. Two deficiencies result in the absence of the same structure from the axoneme of the sperm tail--the dynein-containing outer arm extending from the A subtubule of the peripheral doublet; they also result in the absence from primary spermatocyte nuclei of aggregates of tubuli in one case and reticular material in the other. A third deficiency causes the appearance in the primary spermatocyte of the crystals characteristic of X0 males and the irregular distribution during meiosis of nuclear and cytoplasmic elements to the spermatids. The fourth deficiency results in the misalignment of the developing axoneme with the mitochondrial derivatives and is first detectable in the onion nebenkern stage of the spermatid. Finally for two deficiencies the first abnormalities detected were during later stages and comprise a syndrome found in most of the steriles. We attribute this phenotype to the indirect effects of earlier lesions.

Morphogenesis of mitochondria in male germ cells in cultivated cytocysts begins in early prophase I at which time mitochondria thicken and become ordered along the spindle apparatus during meiosis. At the end of the second meiotic division they aggregate to form the Nebenkern. In the presence of colchicine or cytochalasin B mitochondria are able to begin differentiation, although the correct course of meiosis is not guaranteed. In medium supplemented with colchicine they undergo normal thickening but do not aggregate, in a pattern known from untreated cultures. This may indicate that microtubules are involved in the aggregation process of mitochondria as colchicine is known to inhibit microtubule formation. Moreover, in cell cultures treated with cytochalasin B mitochondrial aggregation does occur; it is concluded that microfilaments, which are sensitive to cytochalasin B, do not play a detectable role in the aggregation of mitochondria.

This paper reports an electron microscope study of typical and atypical spermatogenesis in the pond snail, Cipangopaludina malteata. In the typical spermatid the nucleus undergoes profound changes as development proceeds, affecting both its form and internal fine structure. A large number of roughly parallel, dense filaments, arranged along the long axis of the nucleus, fuse with each other to form in the end the homogeneous helical body characteristic of the head of the adult spermatozoa. The nebenkern is apparently mitochondrial in nature and, in its early development, is similar to that of insects except that it appears as a double structure from the beginning. As differentiation proceeds, the mitochondria lose their membranes, and the residual, now denuded cristae, reorganize to give a parallel radial arrangement. In the last stages of development, the nebenkern derivations become applied to the sheath of the middle piece in a compact helical fashion. In the development of the atypical spermatozoa, the nucleus fails to differentiate and simply shrinks in volume until only a remnant, devoid of DNA, is left. The cytoplasm shows numerous vesicles containing small Feulgen-positive bodies, 80 to 130 mµ in diameter. These vesicles plus contents increase in number as spermatogenesis proceeds. The "head" structure of the atypical spermatozoa consists of a bundle (7 to 17) of tail flagella, each with a centriole at its anterior end. The end-piece of the atypical form appears brush-like and is made up of the free ends of the several flagella.

A helical structure which had previously been reported in the cytoplasm of many cell types in the desert locust, Schistocerca gregaria, was found in large quantities in the developing male germ cells. The helices were isolated from these cells and negatively stained. The helices, which lie free in the cytoplasm of spermatogonia, primary spermatocytes and secondary spermatocytes (sometimes associated with a second type of helix, a double helix), closely invest the outer surface of the Nebenkern membrane in spermatids. As the Nebenkern elongates, the helices gradually disappear and are replaced by microtubules. The mitochondrial derivatives of the Nebenkern, which form part of the sperm tail, are in late spermatids completely surrounded by a sheath of parallel microtubules.

SYNOPSIS The spermatogenesis of the honey-bee, Apia indica (F.), has been worked out with special reference to the abortive meiosis and the role of cytoplasmic organelles. The haploid number of sixteen chromosomes in the parthenogenetic males remains constant through-out spermatogenesis. Meiosis I is abortive, resulting in the formation of a definite non-nucleated cytoplasmic bud containing some extra-nuclear spindle fibres and the cytoplasmic body. The significance of an extra-and an intra-nuclear spindle in relation to the abortive meiosis is discussed. Meiosis II is normal so far as the nuclear division is concerned. The cytoplasm, however, divides unequally, resulting in the formation of a normal spermatid and a diminutive non-functional spermatid body. The mitochondria are in the form of filaments and granules in the early stages. During meiosis I they assume a characteristic “blebbed” appearance. In the spermatid they fuse to form a typical mitochondrial nebenkern, which ultimately gives rise to the mitochondrial sheath around the axial filament of the sperm. A number of typical duplex spheroids (“Golgi dictyosomes”), each with a lipid sheath enclosing a non-lipid medulla, are present in the spermatocytes. In the spermatid these duplex spheroids fuse to form a typical single acroblast which is sloughed off after depositing a pro-acrosome at the nuclear membrane. The pro-acrosome condenses to form the acrosome of the ripe sperm.

The effect of x-irradiation on grasshopper spermatogenesis was studied with the aid of light and electron microscopy. The insects were irradiated at the second instar prior to the presence of maturation stages and observed at the last instar and imago stages. Dosages of 100 to 600 roentgens were found to retard the differentiation of the nucleus and mitochondrial nebenkern in spermatids. Evidence is presented that irradiation causes a curtailment and disorganization in the differentiation of the nebenkern from mitochondria. The above doses also induced the formation of supernumerary centrioles, flagellar filaments and acrosomes; nuclear disorganization as well as pycnosis and fragmentation also occur. The nucleus appears to be drawn toward each radiation-induced supernumerary acrosome, with consequent multipolarity of the nucleus. Induction of a set of flagellar filaments is seen only where the centriolar structure is in contact with the nucleus. Details are given of an organelle, heretofore not described, that is composed of anastomosed and interwoven cytoplasmic strands.

Eight temperature-sensitive (ts) male sterile mutations have been induced by ethyl methanesulfonate treatment of Y chromosomes derived from a selected temperature-resistant Amherst wild-type stock of Drosophila melanogaster. Males carrying such mutated Y chromosomes (Yts) are sterile when raised at 29°C but fertile when reared at 22°C. Complementation tests of the mutants with Y chromosome fragments, deletions, and inter se localized all eight to the long arm of the chromosome in four different complementation groups. When Yts-bearing males, reared to adulthood at 22°C, were subjected to a 48-hr regimen at 29°C and mated to fresh virgin females daily, a significant reduction in fertility resulted 5 days after initiation of 29°C treatments. This period of sterility was transient (48–72-hr duration) and corresponded to a temperature-sensitive period (TSP) of spermatogenesis during the primary spermatocyte stage. A more precise definition of the TSP utilized exposure of subadult males to 29°C at selected developmental periods during which only certain germ cell stages are present. Upon eclosion adult males were subjected to a similar schedule of consecutive matings of 12-hr duration in order to detect any delay in the appearance of fertility. Different ts males could be distinguished by the resultant pattern of sterility, and the TSP of different mutations thus localized to either primary spermatocyte or immediately post-meiotic stage. Associated with Yts-mediated sterility, spermiogenesis is defective at restrictive temperature as evidenced by the production of nonmotile sperm and a failure to transfer such sperm to the female during copulation. In addition, electron microscopy detected a variety of ultrastructural abnormalities, including defects of axoneme formation, irregularities of Nebenkern derivative development, and failures of separation from the syncitial state or mature cyst with subsequent degeneration.

The present paper reports an electron microscope study of spermatogenesis in Hyriopsis schlegelii, mother mussel of fresh-water pearl in the lake Biwa-ko. In the spermatid the nucleus undergoes profound changes as development proceeds, affect-ing both its form and internal fine structure. A large number of roughly parallel, dense filaments, arranged along the long axis of the nucleus, fuse with each other to form in the end, the homogenous body characteristic of the head of the adult spermatozoa. The nebenkern is. apparently mitochondria) in nature and, in its early development, is similar to that of insects. In the early spermatid, all the mitochondria are collected into a single nebenkern which is. then divided into two spherical bodies. The nebenkern are similar in shape and structure to the original mitochondria and differ only in their large size. In the last stages of develop-ment, the nebenkern divided into five spherical bodies around the axial fibrils of developing-tail flagellum. The axial fibrous components of the tail flagellum is composed of nine peripheral pairs of filaments and a pair of central filaments.

Small pieces of the sperm sacs of Lumbricus herculeus were fixed for 4 hours in chrome-osmium, embedded in methacrylate, sectioned with a Porter-Blum microtome, and studied with a R.C.A. EMU-2C electron microscope. Each spermatid of a group developing synchronously is attached by a cytoplasmic strand to a common nutrient protoplasmic mass. This mass contains mitochondria and yolk bodies but is anucleate. The proximal centriole, that is, the centriole nearer the nucleus, is at first associated with a small peg which becomes firmly attached to the nuclear membrane. Later these two bodies become separated during the development of the middle-piece which is differentiated in the usual manner from a nebenkern formed by the fusion of 6 or 7 mitochondria. The acrosome develops in relation to the dictyosome (Golgi body), itself composed of 8 or more individual flattened sacs and situated in the cytoplasm opposite the point of attachment of the spermatid to the nutrient mass. Soon after its formation, the acrosome becomes incorporated into a cytoplasmic appendage or acrosome carrier. The carrier moves from its original position, along the lateral border of the elongating nucleus, to the distal margin of the nucleus where the acrosome is deposited. No evidence was found of a centriole located at the point of junction between nucleus and acrosome as suggested by earlier workers.

1. Observations on the morphological changes of mitochondria preparatory to the formation of the nebenkern, as well as changes within the nebenkern, are reported. 2. Mitochondria enlarge and divide during the meiotic divisions. 3. The mitochondria of the spermatid elongate, become filamentous, form a crescent, and partially encircle the nucleus. 4. Nodes which develop on either end of the crescent become entwined as they move toward each other. 5. The two nodes coalesce to form a filamentous or early type nebenkern which is described by others as chromophilic. 6. Internal rearrangement and partial dissolution of the filaments result in the development of the late or chromophobic nebenkern which separates into two distinct bodies. 7. The nebenkern moieties send out processes toward the centrosome, and after making contact, elongate and occupy part of the space between the tail filaments and sheath of the spermatozoon.

A morphogenetic process that transforms spermatids from a syncytial state to a state in which each spermatid is invested in its own membrane, is initiated at the head region of the spermatid bundle and traverses through the entire length of the bundle in the testis of Drosophila melanogaster. This process not only eliminates the syncytial bridges between spermatids but also removes unneeded organelles and the excess parts of the nuclear membrane, nucleoplasm and cytoplasm. It also brings about structural modifications to flagellar elements. The propagation of this process is seen as the caudal movement of a fusiform swelling of the spermatid bundle, 100 μ or more in length. Spermatids are individualized in the basal half of the swelling, whereas they remain syncytial in the apical half. The swelling increases its volume as it accumulates cytoplasmic debris while traversing the sperm bundle, from about 15 μ in maximum diameter in the basal testicular region to as large as 30 μ at the apical end where it becomes a bag of wastes. A variation of the process in a mutant stock which is known to inactivate up to half of the products of meiosis is briefly described. The morphological change of interspermatid bridges prior to the individualization is also reported.

The fine structure of the mature spermatozoon of the insect Thermobia domestica has been investigated. This flagellate spermatozoon is unique with respect to the relative positions of the centriole, nucleus, mitochondrial nebenkern derivatives, and acrosome along the length of the cell. The acrosome lies at the posterior end of the nucleus. Unlike spermatozoa of most animals, the Thermobia spermatozoon has a nucleus that reveals an unusual lamellar pattern of the material inside of it. This flagellate spermatozoon is also unusual in its tendency to intertwine with other spermatozoa, and during movement the intertwined portion of two (double) spermatozoa is always directed forwards. In the axial filament complex, one of the fibrils of each peripheral double fibril bears a hook-shaped process. An indistinct granular material is seen between the 9 outer coarse fibers. Cytoplasmic tubules, probably corresponding to the "microtubules" of other investigators (67), have been observed around the nucleus, mitochondrial nebenkern derivatives, axial filament complex, and acrosome. A description is given of a complex membrane system which surrounds and separates the main organelles of the cell from each other.

Drosophila parkin, the ortholog of the human parkin gene, responsible for a familiar form of autosomal recessive juvenile parkinsonism, has been shown previously to be involved in Drosophila male fertility. Loss-of-function mutations in the parkin gene cause failure of spermatid individualization by affecting the proper progression of the actin-based investment cones that assemble in the nuclear region, but fail to translocate in synchrony down the cyst. In parkin mutants, the investment cones are scattered along the post-elongated spermatid bundles and fail to act properly in the process of sperm individualization. Using phase-contrast and electron microscopy analysis, we demonstrate that the parkin spermatids assemble a seemingly normal onion-stage nebenkern, but when the axoneme elongates only one mitochondrial derivative unfurls from the nebenkern. This unique mitochondrial derivative undergoes abnormal shaping and condensation during spermatid elongation. Our results indicate that parkin gene function is necessary for mitochondrial morphogenesis during earlier and later phases of spermiogenesis. The failure of cyst individualization may be due to the sensitivity of investment cone movement to the perturbation of mitochondrial morphology during spermatid elongation.

Sperm development has been examined in vinblastine-treated imagos of Drosophila melanogaster. In treated flies, most cytoplasmic microtubules of the spermatids were absent with the exception of the cross-linked nuclear-sheath microtubules. Spermatids that had these cross-linked microtubules expressed variable degrees of normal nuclear differentiation which could be correlated with the numbers of cross-linked microtubules. In addition to nuclear effects, axonemes of vinblastine-treated spermatids did not elongate and the Nebenkern derivatives showed highly aberrant profiles. Based on these observations, cytoplasmic microtubules appear to be prerequisite subcellular elements for the normal differentiation of sperm in Drosophila melanogaster. Additionally, the data from this study suggest that there are two distinct functional classes of cytoplasmic microtubules in spermatids of Drosophila melanogaster, one involved in nuclear differentiation and the other in the generation of the flagellum. Furthermore, it is proposed that the nuclear-sheath microtubules function as subcellular mediators of nuclear shape change.

In elongating spermatids of Drosophila melanogaster , the axoneme is believed to grow by the addition of microtubule subunits at the caudal end, which is enclosed within a short cilium. In fact, the anterior region of tail develops ahead of the posterior region in the structure of axoneme as well as in its relation with the mitochondrial derivatives. The angular relation between the axoneme and the derivatives is not fixed in a short region near the caudal tips of the derivatives. An important part of the mechanism of fixing the relation along the enormously long tail could be the progressive “zipping” of the organelle by following the relation established in the anterior region. The furrow, which divides the surface of the “onion” nebenkern into equal halves belonging to the two subunits, is in alignment with the axoneme. The bisection of the surface, which would provide the subunits with an equal opportunity to associate with the axoneme at the furrow, seems to be an important functional feature of the nebenkern formation. The initial interlocking and the subsequent, gradual unlocking of the subunits may also serve the purpose of assuring the precise positioning of the subunits along the growing axoneme. During the transformation of the 7- μ m wide nebenkern into the 1800- μ m long mitochondrial derivatives, its volume is approximately preserved but its surface area is greatly enlarged. The sum of the surface areas of the shells of the onion amounts to about 30% of the total surface area of the fully elongated derivatives, suggesting another function of the nebenkern formation to be the storage of surface area in the form of concentric shells. Spheroidal bodies 0.5 μ m or less wide, which are elaborate extensions of the outer mitochondrial membrane, are likely sites for the synthesis of the additional area of the outer membrane. For the inner membrane, the possibility of the cristae serving as a reservoir is suggested. Similar but modified mechanisms are expected to be found in different arthropods.

The ultrastructural analysis of the male sterile mutant ms (1) 413 revealed that (a) spermatid differentiation is independent of meiosis; the spermatocytes of this mutant do not divide but develop directly into spermatids having one nucleus and four axonemes. (b) Ms (1) 413 is a timing mutation in which the mitochondria cluster prematurely in the primary spermatocyte but do not form a Nebenkern in the spermatid. (c) Fusion of mitochondria into a Nebenkern is not essential for mitochondrial differentiation in the spermatid; in the ms (1) 413 mutant the mitochondria do not fuse but nevertheless differentiate similarly to the mitochondrial derivatives of the wild type. (d) The differentiation of the nuclear envelope is, apparently, abnormal, as indicated by the following phenomena: lack of chromosomal condensation and spindle formation in the spermatocytes; highly irregular distribution of nuclear envelope annuli, condensed chromatin and perinuclear microtubules in the spermatids; and lack of connection between basal bodies and spermatid nucleus.

The ultrastructure of spermatid differentiation in an autosomal male sterile mutant of Drosophila melanogaster is described. The mutant ms(2)3R is characterized by failure of the cytokinetic divisions which normally accompany meiosis. The primary spermatocyte undergoes nuclear division, but a failure of cytokinesis leaves 4 spermatids to develop within a common cytoplasm. The mitochondria fuse, usually forming a single large nebenkern, which then divides into two approximately equal parts as does a normal nebenkern. In the mutant, these two mitochondrial derivatives usually undergo further division generally giving rise to 8 or fewer mitochondrial derivatives. Multiple paracrystalline bodies are often observed in the primary mitochondrial derivatives. Up to 4 paracrystalline bodies may be observed in a single section of a derivative, one at each contact point between the membranes of the primary mitochondrial derivatives and the membranes around the 4 axonemes contained in the common cytoplasmic unit. The units of four spermatids almost complete maturation before the bundles degenerate. It appears that, with the exception of the nebenkern and its derivatives, cytoplasmic separation is not a prerequisite for the normal differentiation of most spermatid organelles.

1. Although mitochondria can not be definitely demonstrated in the primary spermatogonia of Passalus cornutus, they are present in the secondary spermatogonia in the form of numerous and diffusely spread granules.2. The mitochondria increase in number during the growth period, and in the later stages are found in the form of threads (chondrioconts) which lie in the direction of the chief axis of the cell.3. During the maturation divisions, the mitochondria envelop the spindle peripherally and are divided by the cell constrictions, so that daughter cells receive approximately equal amounts.4. The mitochondria of the spermatid form the Nebenkern, which later is pierced by the axial filament. As the latter grows, the Nebenkern elongates, forming a sheath about it.5. Spindle remains are found forming connections between the spermatogonia. The spindle remains of the last spermatogonial division persist throughout the entire growth period of the spermatocyte.6. A spindle derivative is found in the spermatid, a ...