Search

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.

Wolbachia is a group of intracellular symbiotic bacteria that widely infect arthropods and nematodes. Wolbachia infection can regulate host reproduction with the most common phenotype in insects being cytoplasmic incompatibility (CI), which results in embryonic lethality when uninfected eggs fertilized with sperms from infected males. This suggests that CI-induced defects are mainly in paternal side. However, whether Wolbachia-induced metabolic changes play a role in the mechanism of paternal-linked defects in embryonic development is not known. In the current study, we first use untargeted metabolomics method with LC-MS to explore how Wolbachia infection influences the metabolite profiling of the insect hosts. The untargeted metabolomics revealed 414 potential differential metabolites between Wolbachia-infected and uninfected 1-day-old (1d) male flies. Most of the differential metabolites were significantly up-regulated due to Wolbachia infection. Thirty-four metabolic pathways such as carbohydrate, lipid and amino acid, and vitamin and cofactor metabolism were affected by Wolbachia infection. Then, we applied targeted metabolomics analysis with GC-MS and showed that Wolbachia infection resulted in an increased energy expenditure of the host by regulating glycometabolism and fatty acid catabolism, which was compensated by increased food uptake. Furthermore, overexpressing two acyl-CoA catabolism related genes, Dbi (coding for diazepam-binding inhibitor) or Mcad (coding for medium-chain acyl-CoA dehydrogenase), ubiquitously or specially in testes caused significantly decreased paternal-effect egg hatch rate. Oxidative stress and abnormal mitochondria induced by Wolbachia infection disrupted the formation of sperm nebenkern. These findings provide new insights into mechanisms of Wolbachia-induced paternal defects from metabolic phenotypes., Author summary Wolbachia are among the most successful intracellular bacteria that can infect many arthropod species as well as filarial nematodes. Wolbachia can manipulate the reproduction of their insect hosts to enhance their transmission through host populations. Cytoplasmic incompatibility (CI) is the most common phenotype induced by Wolbachia in insect hosts, which results in embryonic mortality when Wolbachia infected males cross with normal (uninfected) females. This indicates that the impact of Wolbachia infection is mainly on paternal side. Evidence has suggested that the metabolism is strongly linked with male reproduction. We were interested in investigating whether Wolbachia-induced metabolic changes involve in the mechanism of paternal defects in embryogenesis. Here, using comparative metabolomics method, we identified 414 potential differential metabolites related to 34 metabolic pathways between Wolbachia-bearing and Wolbachia-free male Drosophila melanogaster. Then, by targeted metabolomics analysis, gene overexpression, and transmission electron microscopy (TEM) techniques, we found that in Wolbachia infected D. melanogaster, energy metabolism and oxidative stress were enhanced, and the development of mitochondria-derived nebenkern during spermatogenesis was impaired. These may be the main causes of paternal defects induced by Wolbachia.

Mitochondria, usually ovoid structures no larger than 0.5 microns can fuse into structures that are 5 microns and larger such as nebenkern, spheroidal or cup-shaped mitochondria, and even mega-mitochondria. In studying differentiation of human endometrial epithelial cells, it became clear that formation of mitochondrial superstructures was an essential part of the process. In this paper, the origins, function, and demise of these superstructures called mitonucleons are described. In the course of reading papers about mitochondrial superstructures, it became obvious that there are important similarities, particularly with regard to function, between the mitonucleon and the nebenkern, a superstructure essential for dramatic tail elongation during spermatogenesis in grasshoppers, drosophila, and other insects. Close inspection of photomicrographs of differentiating mitonucleons during the first 12 hours suggests that gases build up in vacuoles within the mitochondrial superstructure creating pressure that elevates syncytial membranes and compresses nuclear aggregates contained within the mitonucleon.

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 southern cowpea weevil, Callosobruchus maculatus (F) is a severe agriculture pest worldwide. In the current work, newly emerged adult males of C. maculatus have been irradiated with a low dose of 20Gy gamma radiation. The inherited deleterious effects on the fecundity, hatchability, adult emergence, and the sterility percent were recorded for the progenies F1 and F2 of the irradiated parental males. The fecundity, hatched larvae, the number of males and females were reduced in both F1 and F2. The sterility percent was high in F1 (70.8%) and increased in the F2 (88.3%) generation. Histopathological effects were also documented in the testes of F1 and F2 progenies. The spermatids and sperms have exhibited a variety of abnormalities. In the early spermatids, the nebenkern outer cell membrane was ruptured. The spermatid nucleus loses its homogeneous texture and has multiple foci of dense chromatin, as well as, profiles range has little dense material. In some groups, the nucleus had a peculiar ring of chromatin. The sperms had shown a variety of aberrations. The sperms irregularity distributed in lysed cysts by unusual manner. Also, some sperms had remarkably enlarged axoneme and small rounded nucleus. Many of the sperm cells were observed with two axonemes, abnormal mitochondria derivatives, and more than two accessory bodies. These results indicate that the low dose of 20 Gy induces semi-sterility in C. maculatus through generations. The same technique would help to improve using of sterile insect technique for other agriculture pests.

Ultrastructure of spermatozoa in fairy shrimp Streptocephalus dichotomus revealed that they are amoeboid type with no acrosome and flagella. Surface topography of the spermatozoon is smooth with occasional pseudopodial projections. Transmission electron micrographs of spermatozoa show organelle and the mitochondria which is not fused to form the so called ‘Nebenkern’. The testicular lumen reveals spermatozoa in varying sizes and shapes.

A study on the Nezara viridula male gonad cells was undertaken to compare normal development and development of insect cells after irradiation of 4th instar nymphs with a dose of 40 Gray. Visually, morphologically, biochemically and cytologically the insects were not all uniformly affected by the radiation. In all aspects of development there was a range from severely affected to nearly normal. Irradiated insects appeared to move slowly and were unable to mate with non-irradiated females. The testes of the males varied from bright orange to grey in colour and all were smaller in size than non-irradiated testes. The ultrastructure of the developing sperm showed abnormalities the axonemes, the mitochrondrial derivatives, nebenkern and centrioles. Cytochemically, the main difference observed was the presence of granules heavily stained with acid phosphatase in between mitochrondrial derivatives. The chromosomes of these irradiated insects were highly fragmented. Although a few sperm in irradiated insects appeared normal no progeny were produced as insect did not mate. The sterile insect technique (SIT) requires a balance between the effective radiation dose to achieve partial or full sterility, while maintaining physical fitness. The observation that abnormalities varied from almost none to severe at 40Gy could help the development of SIT for control of N. viridula and other Pentatomidae.

We studied the fine structure of some classical and six variant mitochondria from different tissues viz. proboscis gland, spinal gland, ovary, testis, and muscle of a fish ectoparasite, Argulus bengalensis. In the proboscis gland and spinal gland, mitochondria are protected within vesicle to preserve their structure and activity from exposure to glandular synthesis for its parasitic mode of feeding. In the oocytes, mitochondria are larger and cylindrical in appearance. Oocyte mitochondria are highly dynamic and exhibit frequent fission and fusion. Those are clustered in the cytoplasm of previtellogenic oocytes which prepare for different synthetic activities for successful reproductive investment. In contrast, mitochondrial abundance is less in the male gametic lineage. The spermatocytes and the nurse cells in the testis have an unusual type of mitochondria, nebenkern which is formed by the fusions of number of mitochondria. A completely different type of mitochondrion is discovered in the flagellum of the spermatozoa. It is provided with fifteen numbers of singlet microtubules at its outer periphery which is a salient feature of the flagellum of this Branchiuran genus. This unique mitochondrion uses the microtubule tract for its movement to distribute energy efficiently along the axoneme. Such mitochondrion and microtubular association provide evidence in favor of phylogenetic relationship between Argulus and pentastomid Raillietiella. In striated muscle of thoracic appendages, mitochondria maintain tight junctions with the endoplasmic reticulum and remain in close apposition of the myofibrils which helps in Ca2+ uptake for stimulating continuous muscular activity required for ventilation of respiratory structures of the parasites.

A coleopteran insect, Trachyderma hispida, was collected from the proximity of ceramic factories in Khorshed district, Alexandria, Egypt, to elucidate the testicular damage elicited by heavy metal pollution. Bioaccumulation of heavy metals was estimated by using X-ray microanalysis. Histological and ultrastructure examination revealed various changes in germ cells in insects collected from the polluted region. The inducible histological alterations included: occlusion and collapsed follicles, disrupted cyst wall and necrotic spermatogenic elements. Ultrastructure changes implied abnormal chromatin condensation, ill-defined nuclear membrane, vacuolated cytoplasm, disintegrated nebenkern, deterioration of mitochondrial derivatives and degeneration of axonemes. In addition, agglutinated spermatids, and sperms with a double tail were noticed. The most noteworthy feature in spermatogonia and spermatocyte was the intra-tubular inclusion bodies. Histological and electron micrographs pointed out the structure of the parietal cell which functioned as a Sertoli cell. Adverse damage to these cells by ceramic pollution was reported. In conclusion, by using insects as bioindicators, ceramic production may impair reproduction.

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.

Mitochondria morphogenesis during spermatogenesis in Drosophila carrying merlin gene mutations was examined by electron and fluorescent microscopy. Hypomorphic allele mer3; null allele mer4; and genetic construct Mer +, which encodes full-size protein, were applied in the experiments. A detailed analysis of anomalies in spermatogenesis induced by these mutations shows that Merlin is important for the formation, structural support, and modification of the apparatus of mitochondria (nebenkern). The possible role of a Merlin protein as an adaptor protein that can link mitochondria with the cytoskeleton and its activity defined by molecule conformation are discussed.

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.

An important property of the endoplasmic reticulum (ER) is its ability to change morphology and intracellular localization during the cell cycle and differentiation. Visualization of the ER membranes using the protein disulphide isomerase (PDI) GFP chimeric protein makes it possible to trace the dynamics of all these processes and expose transitional forms and intermediate configurations. In this article the results of the study of the ER morphology during spermatogenesis of D. melanogaster are presented. It was shown that ER membranes retain high level of GFP-fluorescence through all stages of spermatogenesis, so that revealing of the stage-specific features of the ER organization was possible. The ER network has distinctive reticular morphology during the interphase and early prophase. Right before the cell division, this morphology changes and ER forms a system of branchless filamentous membranes. In prometaphase, these membranes form concentric circles adjacent to the nuclear membrane; from metaphase to telophase, they lengthen along the axis of cell division and resemble a spindle. Later, in the next interphase, this configuration transforms to a reticular structure seen previously. At the beginning of spermatid differentiation, the ER encompasses nebenkern and nucleus and elongates adjacent to them. During the latest stages of spermatogenesis, the ER network dissociates into separate membranous granules that are eliminated from the cyst with the individualization complex. Possible mechanisms of the ER dynamics and reorganization are discussed.

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.

The genes don juan (dj) and don juan like (djl) encode basic proteins expressed in the male germline. Both proteins show a similar expression pattern being localized in the sperm heads during chromatin condensation and along the flagella. Prematurely expressed Don Juan–eGFP and Myc-Don Juan Like localize to the cytoplasm of spermatocytes and in mitochondrial derivatives from the nebenkern stage onward suggesting that both proteins associate with the mitochondria along the flagella in elongated spermatids. Premature expression of Myc-Don Juan Like does not impair spermatogenesis where-as Don Juan-eGFP when prematurely expressed causes male sterility as spermatids fail to individualize. In spite of the sequence identity of 72% on the nucleotide level and 42% on the protein level, the presumptive promoter regions and the untranslated regions of the mRNA are diverged. Our in vivo analysis revealed that don juan and don juan like are transcriptionally and translationally controlled by distinct short cis regulatory regions. Transcription of don juan and don juan like depends on the male germ line specific TAFII80, Cannonball (Can). Translational repression elements for both mRNAs are localized in the 5′ UTR and are capable to form distinct secondary structures in close proximity to the translational initiation codon. Developmental Dynamics 235:1053–1064, 2006. © 2006 Wiley-Liss, Inc.

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.

The Merlin gene of Drosophila is homologous to the human Neurofibromatosis 2 (NF2) gene, an important regulator of proliferation and endocytosis of cell receptors. It was earlier shown that the Thr559 residue of the Drosophila Merlin protein was homologous to Ser518 of the human protein (which was already known to undergo phosphorylation); hence, it was assumed that Thr559 of Drosophila also was a substrate of phosphorylation. The mutant Merlin proteins MerT559D (an analog of the phosphorylated form) and MerT559A (a nonphosphorylated form) were constructed and tested, under the conditions of ectopic expression, for the ability to correct the spermatogenesis defects induced by the Mer4 mutation. The mutant form MerT559D was demonstrated to restore the abnormal nebenkern phenotype induced by this mutation, whereas the MerT559A substituted form did not restore this phenotype. Ectopic expression o the wild-type Merlin protein, MerT559A mutant form, and mycMer345–635 truncated protein in a normal genotype resulted in the abnormal nebenkern phenotype, whereas this phenotype was not observed in the case of ectopic expression of the MerT559D analog of the phosphorylated form. Ectopic expression of the mycMer3, mycMerΔBB, and mycMer1–379 truncate variants led to disturbance of meiotic cytokinesis.

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.

Meiosis in higher organisms is programmed as a part of gametogenesis. It should be discussed separately from a yeast meiosis that initiates in response to extracellular nutrient conditions. In this chapter, we would like to introduce current knowledge including our new findings about dynamics of meiosis in Drosophila males. At first, we will introduce general views of cell divisions and cell growth during Drosophila spermatogenesis as we illustrated in Fig. 1A. At the tip of the testis in adult Drosophila males, several germline stem cells (GSCs) are surrounding next to a cluster of smaller hub cells. The GSCs receive a signal to maintain their multi-potential stem cell characteristics secreted from the adjoining hub cells. A secreted protein encoded by the unpaired gene acts as a ligand of the maintenance signal (Kiger, et al., 2001, Fuller & Spradling, 2007). The insulin-like peptides in hemolymph first induce the cell cycle progression of the male GSCs from G2 phase to M phase (Ueishi et al., 2009). Between two daughter cells derived from asymmetric division of the GSC, a proximal daughter cell exclusively receives the Unpaird signal and becomes a self-renewed GSC. The other distal daughter cell leaves the niche and begins to differentiate as a spermatogonium. The spermatogonium then undergoes four cell cycles and generates a 16 cell unit known as a cyst (as a review Fuller, 1993). In ever mitosis, all spermatogonia within a cyst undergo cell divisions synchronously. Cytokinesis in spermatogonia mitoses as well as in following meiotic divisions terminates incompletely. The cleavage furrow ingression is arrested at the middle of cytokinesis and then the contractile rings transform into cytoplasmic bridges called ring canals (Hime et al., 1996). These 16 spermatocytes synchronously enter a growth phase during which the cells remarkably increase in volume by up to 25 times. Following completion of the enormous cell growth, primary spermatocytes carry out two consecutive meiotic divisions. A cyst of 16 spermatocytes gives rise to 64 spermatids simultaneously as a consequence of meiotic divisions. Every spermatid in a cyst at onion stage contains equally sized nucleus and Nebenkern that is a single large aggregate derived from mitochondria. This should be achieved as a consequence of proper chromosome segregation and cytokinesis in germ line cells as well as equal partition of mitochondria (Castrillon et al., 1993, Ichihara et al., 2007).

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.

By use of osmium ferricyanide (OsFeCN) staining the fate of cytoplasmic membranes was followed during spermiogenesis in the cotton seed bug (Dysdercus intermedius). During early spermiogenesis interzonal lamellae of endoplasmic reticulum become aggregated as a stack of membranes traversing the entire cell body from the nucleus to the cytoplasmic bridge connecting neighbouring spermatids. Cisternae of endoplasmic reticulum ensheath the acroblast from which vesicles of different sizes are pinched off into the cytoplasm. The oxalate method was used to show that acroblast and associated vesicles are calcium-sequestering sites in spermatids. Membrane profiles with dense calcium oxalate precipitate derived from the acroblast form an uninterrupted membranous sheath at the apical side of the nucleus where the proacrosome will be attached. With further development of the spermatids, the vesicles derived from the acroblast also participate in forming a calciumsequestering sheath enveloping the axoneme and the mitochondrial “nebenkern” derivatives.

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...

We constructed balancer-chromosomes for the large autosomes ofDrosophila hydei and screened more than 16000 chromosomes for male sterile mutations in order to dissect spermatogenesis genetically. 365 mutants on the X chromosome and the autosomes 2, 3, and 4 were recovered and analysed cytologically in squash preparations under phase-contrast optics. The majority of the mutations allows a rather advanced differentiation of the spermatozoa. At the light-microscopical level, it is possible to classify these mutations with respect to individualization, coiling or motility of the mutant spermatozoa. In contrast, a small number of mutants exhibits conspicuous, pleiotropic phenotypes. Gonial divisions, the shaping of the spermatocyte nucleus and male meiotic divisions are controlled by X chromosomal or autosomal genes which can mutate to male sterile alleles. A number of nonallelic 3rd chromosome male sterile mutations interfere with the unfolding of the Y chromosomal lampbrush loops. Other autosomal male sterile mutations modify the morphology of these lampbrush loops. Another group of mutations inhibits the formation of the nebenkern while the development of the spermatid nucleus and the flagellum can proceed. Such male sterile mutations can decouple the development of nucleus, protein body, nebenkern, and flagellum of the spermatid. Thus, we can describe spermatogenesis inDrosophila as the coordinate execution of the individual developmental programs of the different components of the spermatozoon.

Spermatogenesis in Drosophila melanogaster serves as an excellent model system for the isolation and analysis of genes required in the control of chromosome segregation and cytokinesis. We report here the isolation and molecular characterization of a novel P-element induced allele of the des-1 gene, which leads to male sterility as a consequence of the failure of central spindle assembly in meiotic spermatocytes and the formation of aberrant meiotic end products characteristic of cytokinesis failure. We have raised affinity-purified antibodies against a Des-1 fusion protein, and localized the Des-1 protein in Drosophila spermatocytes. We show that the Des-1 protein is colocalized with mitochondria throughout male meiosis, becoming intimately associated with mitochondria along the spindle apparatus during anaphase and telophase, and with the Nebenkern, or mitochondrial derivative, of the meiotic end products. In addition, a significant association of Des-1 with the contractile ring is observed during anaphase and telophase of meiosis. These observations, together with the presence of six potential transmembrane domains in the Des-1 protein, raise the possibility that Des-1 may act as part of an anchoring mechanism that links membrane-bounded cellular compartments to components of the cytoskeleton.

This study compares the sperm morphology of 2 hermaphroditic (Diploria strigosa, Montastrea annularis) and 2 gonochoric (Siderastrea siderea, Dendrogyra cylindrus) Caribbean reef corals. The spermatozoa of the gonochoric species are morphologically more specialized than those of the hermaphrodites. The cytological differences are apparent in the formation of a Spitzenkorper and pro-acrosomal vesicles, a mitochondrial mass (Nebenkern) and a large lipid vesicle in the gonochorists studied. Harrison (1985) first demonstrated that sperm morphology is correlated with sexual reproductive pattern and systematic position in scleractinian corals, and this is supported by the results of this study. The possible functional and phylogenetic bases of sperm morphology are discussed and a brief outline is given of further studies required.

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.

During the development of a sterile male control method for Dermestes frischii Kugelann males were exposed to an X-ray dose of 3.0 krad. The treated testis follicles were investigated using light and electron microscopy. Abnormalities in spermatids and sperm were observed only in cells treated as primary or secondary spermatocytes. Malformed nuclei, nebenkern and acrosomes were detected. Cells frequently failed to start or complete the first or second meiotic divisions. However, spermiogenesis was not inhibited and this resulted in the formation of pseudospermatids of which three types were identified. Only one of these has been previously described. Pseudospermatids were associated with two, three or four sets of quite normal tail organelles. The presence of multiple organelles is discussed in relation to similar findings in other insects.

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.

An electron-microscope study of spermiogenesis and the ultrastructure of mature sperm was made on Eurygaster integriceps. During spermiogenesis, a manchette consisting of two large groups of microtubules and an unusual centriolar adjunct are formed. The latter looks like two half cylinders located almost at right angles to one another. Its wall consists of several dark layers divided by lighter areas. The centriole and its adjunct are not identified in the mature sperm. Bug spermatids have a large amount of amorphous pericentriolar matter, which assists in establishing an unusual nuclear pattern. The mature sperm is distinguished by a number of unique features. Its nucleus consists of three interconnected parts: the inner and outer cylinders and a part freely suspended along the middle piece. The intranuclear channel is blindly closed at the apical end and filled with dark amorphous matter that originates from the pericentriolar matter. The acrosome has an extracellular part resembling a diagonally striated rod, which is sometimes disengaged from its surface. The axoneme has 9+9(2) + 2 tubules. It is connected with the Nebenkern by dark arms.

The sperm of the lovebug, Plecia nearctica possesses an acrosome, which caps the apical and anterior lateral surfaces of a spear-shaped nucleus, which has parallel, bilateral concavities running throughout its length. At the junction of the nucleus and axial filament, a centriole adjunct has developed. An extension of this complex extends along one side of the sperm tail, partially surrounding the nebenkern and axial filament. A single nebenkern is found parallel with the axial filament along most of its length. A 9 + 0 tubule pattern exists in the axial filament with the central pair of tubules being replaced by a prominent central sheath.

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.

When spermiogenesis of Eurygaster integriceps occurs, the pattern of the round nucleus of the spermaiid changes under the influence of a manchette consisting of two large groups of microtubules (up to 130–140 in each). The pericentriolar matter was found to be involved in the changes in the nuclear pattern; it takes part in the formation of the asymmetric intranuclear channel, open at one side. Two zones appear in the nucleus that differ greatly in their rates of chromatin condensation. The two main parts of the future nucleus are thereby established: the apical one with a greater rate of chromatin condensation, which gives origin to the two-walled cylinder, and the outer part, which grows for a long time and extends caudally to the middle piece. At the later stages of nucleus formation, when its larger part is separated from the surface of the middle piece, the vesicles of the endoplasmic reticulum that penetrate between the Nebenkern and the larger part of the nucleus, play a certain role.

When males of the Migratory locust are raised under infra-optimal temperature conditions, namely, with a rythm of 20°C night/28°C day instead of 20°C/33°C, they become sterile. The size of the testis is normal, gonial and meiotic divisions take place, and spermiogenesis is initiated. No ripe sperms are formed, however, and the spermiducts and seminal vesicles remain empty. It is demonstrated that it is spermiogenesis that is altered. The cells of a given cyst do not evolve synchronously as they should do and, in each individual cell, the highly ordered pattern of organelle differentiation is also disturbed. Almost all specialized organelles of germ cells are formed but some develop incompletely (axonemes missing their two central tubules), others are morphologically different from normal (nebenkern with very dense matrix), others are produced in extra copies (mitochondrial derivatives, flagella and centriolar adjuncts). The normal correlations between the states of development of the various organelles are disturbed and the topographical relationships also are distorted even when the individual organelles are morphologically normal. As a result, the spermatids never elongate properly, they never align, and they never form a bundle of ripe spermatozoa.

The ultrastructural damage to the spermatid organelles of Locusta migratoria was studied after one injection of 10 microliter isopropoxy phosphine at 5th instar stage. The effects were prevalent after three to four weeks. Vacuolation was observed in nuclei of early spermatids. Some nuclei of advanced spermatids had crinckled nuclear wall. Occasionally, the nebenkern and its derivatives showed vacuolation. The formation of supernumerary flagellar complexes also occurred in some spermatids. Sometimes, the flagellar filaments were disorganized. The results are more or less similar to those which followed x-ray irradiation and corpora allata implantation.

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.

SYNOPSIS The sperm of Petrobius maritimus is a normal flagellate type and is formed in the normal manner. The acrosome is a clearly marked and easily followed structure. In the final stages the acrosome can be seen to be deposited on the proximal end of the nucleus as a composite structure with a small granule at the tip. It is a secretory product of the Golgi body and not the Golgi body itself. The spent acroblast (dictyosome, Golgi body) drifts down the lengthening spermatid as the acroblast reject, as normally occurs in flagellate spermiogenesis. The centriole region probably consists of the centriole plus the centriole adjunct, but is too small to be resolved by the light microscope. The mitochondrial nebenkern is formed by the aggregation, but apparently not the agglutination, of the mitochondria, and is not typical of the mitochondrial nebenkern of holometabolous insects.