Formation of Phosphoglycosides in Caenorhabditis elegans: A Novel Biotransformation Pathway

Background

Caenorhabditis elegans (C. elegans) has become a widely used model to explore the effect of food constituents on health as well as on life-span extension. The results imply that besides essential nutrients several flavonoids are able to impact the aging process. What is less investigated is the bioavailability and biotransformation of these compounds in C. elegans. In the present study, we focused on the soy isoflavone genistein and its metabolism in the nematode as a basis for assessing whether this model system mimics the mammalian condition.

Principal Findings

C. elegans was exposed to 100 µM genistein for 48 hours. The worm homogenate was extracted and analyzed by liquid chromatography (LC). 11 metabolites of genistein were detected and characterized using LC electrospray ionization mass spectrometry. All genistein metabolites formed by C. elegans were found to be sugar conjugates, primarily genistein-O-glucosides. The dominant metabolite was identified as genistein-7-O-phosphoglucoside. Further interesting metabolites include two genistein-di-O-glycosides, a genistein-O-disaccharide as well as a genistein-O-phosphodisaccharide.

Conclusions/Significance

Our study provides evidence for a novel biotransformation pathway in C. elegans leading to conjugative metabolites which are not known for mammals. The metabolism of genistein in mammals and in C. elegans differs widely which may greatly impact the bioactivity. These differences need to be appropriately taken into consideration when C. elegans is used as a model to assess possible health or aging effects.     

Spermiogenesis and the ultrastructure of mature sperm in Eurygaster intergriceps

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.     

Spermiogenesis in the Australian cockatiel Nymphicus hollandicus

Information on the ultrastructure of parrot spermatids and spermatozoa is limited to only four species with no comprehensive study of spermiogenesis conducted within the order Psittaciformes. The present study was undertaken to describe the development of the cockatiel spermatid using electron microscopy. Four phases of spermatid maturation were documented on the basis of nuclear morphology, development of the acrosome, perforatorium, and axial filament. These phases included 1) round nuclei, 2) irregular nuclei, 3) elongated nuclei with granular chromatin, and 4) elongated nuclei with homogenous chromatin. While development of the cockatiel spermatid was comparable to that of other domestic avian species, we have noted the hollow nature of some chromatin granules, an abnormal formation of the axoneme, the absence of the fibrous sheath around the axoneme of the principal piece, and the absence of an annulus. J. Morphol. , 2012. © 2012 Wiley Periodicals, Inc.     

Succinylated Octopamine Ascarosides and a New Pathway of Biogenic Amine Metabolism in Caenorhabditis elegans

The ascarosides, small-molecule signals derived from combinatorial assembly of primary metabolism-derived building blocks, play a central role in Caenorhabditis elegans biology and regulate many aspects of development and behavior in this model organism as well as in other nematodes. Using HPLC-MS/MS-based targeted metabolomics, we identified novel ascarosides incorporating a side chain derived from succinylation of the neurotransmitter octopamine. These compounds, named osas#2, osas#9, and osas#10, are produced predominantly by L1 larvae, where they serve as part of a dispersal signal, whereas these ascarosides are largely absent from the metabolomes of other life stages. Investigating the biogenesis of these octopamine-derived ascarosides, we found that succinylation represents a previously unrecognized pathway of biogenic amine metabolism. At physiological concentrations, the neurotransmitters serotonin, dopamine, and octopamine are converted to a large extent into the corresponding succinates, in addition to the previously described acetates. Chemically, bimodal deactivation of biogenic amines via acetylation and succinylation parallels posttranslational modification of proteins via acetylation and succinylation of l-lysine. Our results reveal a small-molecule connection between neurotransmitter signaling and interorganismal regulation of behavior and suggest that ascaroside biosynthesis is based in part on co-option of degradative biochemical pathways.     

Spermiogenesis in the cestode Corallobothrium solidum Fritsch, 1886 (Proteocephalidea: Corallobothriinae)

Spermiogenesis of Corallobothrium solidum Fritsch 1886, has been investigated by transmission electron microscopy. The zone of differentiation contains the two centrioles, each with one thin root, being orientated in the same plane only when a single intercentriolar body (ICB) appears between them. A median cytoplasmic process (MCP) develops distally to the two flagella, which are of unequal length, get longer and rotate towards the MCP. The nucleus penetrates into the spermatid body after the fusion of both flagella with the MCP has started. Flagellar roots occur occasionally in some spermatids. New for the Eucestoda are the following findings: 1. cortical microtubules (CMs) are arranged in two short parallel rows in one‐axoneme region of some spermatids; 2. the crested body of spermatid consists either of electron‐dense tubular elements and interposes itself between CMs, or it is rather homogeneous and situated more peripherally above one continuous semicircle of CMs. The present results support previous data that the type of spermiogenesis in proteocephalideans resembles mostly that observed in tetraphyllideans (Onchobothriidae and Phyllobothriidae), thus supporting the view of a close phylogenetic relationship of tetraphyllidean and proteocephalidean cestodes.     

Distinct roles of the two VPS33 proteins in the endolysosomal system in Caenorhabditis elegans

Sec1/Munc‐18 (SM) family proteins are essential regulators in intracellular transport in eukaryotic cells. The SM protein Vps33 functions as a core subunit of two tethering complexes, class C core vacuole/endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS) in the endocytic pathway in yeast. Metazoan cells possess two Vps33 proteins, VPS33A and VPS33B, but their precise roles remain unknown. Here, we present a comparative analysis of Caenorhabditis elegans null mutants for these proteins. We found that the vps‐33.1 (VPS33A) mutants exhibited severe defects in both endocytic function and endolysosomal biogenesis in scavenger cells. Furthermore, vps‐33.1 mutations caused endocytosis defects in other tissues, and the loss of maternal and zygotic VPS‐33.1 resulted in embryonic lethality. By contrast, vps‐33.2 mutants were viable but sterile, with terminally arrested spermatocytes. The spermatogenesis phenotype suggests that VPS33.2 is involved in the formation of a sperm‐specific organelle. The endocytosis defect in the vps‐33.1 mutant was not restored by the expression of VPS‐33.2, which indicates that these proteins have nonredundant functions. Together, our data suggest that VPS‐33.1 shares most of the general functions of yeast Vps33 in terms of tethering complexes in the endolysosomal system, whereas VPS‐33.2 has tissue/organelle specific functions in C. elegans.      

Spermiogenesis initiation in Caenorhabditis elegans involves a casein kinase 1 encoded by the spe-6 gene

Immature spermatids from Caenorhabditis elegans are stimulated by an external activation signal to reorganize their membranes and cytoskeleton to form crawling spermatozoa. This rapid maturation, termed spermiogenesis, occurs without any new gene expression. To better understand this signal transduction pathway, we isolated suppressors of a mutation in the spe-27 gene, which is part of the pathway. The suppressors bypass the requirement for spe-27, as well as three other genes that act in this pathway, spe-8, spe-12, and spe-29. Eighteen of the suppressor mutations are new alleles of spe-6, a previously identified gene required for an early stage of spermatogenesis. The original spe-6 mutations are loss-of-function alleles that prevent major sperm protein (MSP) assembly in the fibrous bodies of spermatocytes and arrest development in meiosis. We have isolated the spe-6 gene and find that it encodes a predicted protein-serine/threonine kinase in the casein kinase 1 family. The suppressor mutations appear to be reduction-of-function alleles. We propose a model whereby SPE-6, in addition to its early role in spermatocyte development, inhibits spermiogenesis until the activation signal is received. The activation signal is transduced through SPE-8, SPE-12, SPE-27, and SPE-29 to relieve SPE-6 repression, thus triggering the formation of crawling spermatozoa.     

Spermiogenesis in Coenobita clypeatus,I. Sperm structure

The sperm of the tropical land hermit crab, C. clypeatus, has an elongate acrosome anterior to a lamellar region of cytoplasm. Mitochondria near the lamellar region are associated with microtubules. These microtubules project into the 3 cytoplasmic arms. The nucleus occupies the posterior-most position in the sperm. The chromatin is not condensed and numerous projections of nuclear materials are seen. It is not known how the various organelles of the sperm function during fertilization.     

Morphological characterization of the testicular cells and seminiferous epithelium cycle in six species of Neotropical bats

We know little about the process of spermatogenesis in bats, a great and diverse clade of mammals that presents different reproductive strategies. In the present study, spermatogenesis in six species of Neotropical bats was investigated by light microscopy. On the basis of chromatin condensation, nuclear morphology, relative position to the basal membrane and formation of the flagellum, three types of spermatogonia were recognized: dark type A (A_d), pale type A (A_p), and type B; the development of spermatids was divided into seven steps. With the exception of Myotis nigricans, the seminiferous epithelium cycle of the other five species studied was similar to those of other mammals, showing gradual stages by the tubular morphology method. Asynchrony was observed in the seminiferous epithelium cycle of M. nigricans, shown by overlapping stages and undefined cycles. The frequencies found in the three phases of the cycle were variable with the greatest frequency occurring in the postmeiotic phase (>50%) and the least in the meiotic phase (<10%). The similarities observed in the five species of Phyllostomidae appeared to be related to their phylogenetic relationship and shorter divergence times, whereas the differences in M. nigricans appeared to be related to its greater phylogenetic distance because the Vespertilionidae family diverged earlier. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Ultrastructural features of spermatozoa and their phylogenetic application in Zaprionus (Diptera,Drosophilidae)

The genus Zaprionus consists of approximately 60 species of drosophilids that are native to the Afrotropical region. The phylogenetic position of Zaprionus within the Drosophilidae family is still unresolved. In the present study, ultrastructural features of spermatozoa of 6 species of Zaprionus as well as the species Drosophila willistoni and Scaptodrosophila latifasciaeformis were analyzed. The ultrastructure revealed that the species have the same flagellar ultrastructure. Two mitochondrial derivatives, one larger than the other, close to the axoneme were present, primarily in D. willistoni (subgenus Sophophora). Except for Z. davidi and Z. tuberculatus, the analyzed species had paracrystalline material in both mitochondrial derivatives. Moreover, the testes showed 64 spermatozoa per bundle in all of the species. In the cluster analysis, 6 Zaprionus species were grouped closely, but there were some incongruent positions in the cladogram. The results indicated that sperm ultrastructure is an important tool for elucidating the phylogeny and taxonomy of insects.     

Ultrastructure of spermatogenesis in the short‐tailed fruit bat,Carollia perspicillata (Chiroptera: Phyllostomidae: Carollinae)

Among species of the Chiroptera, spermatogenesis and the fully differentiated spermatozoa differ in morphological and ultrastructural detail. This study therefore aimed to ultrastructurally characterize the spermatogenesis and the spermatozoa of Carollia perspicillata (Phyllostomidae) and compare the process with other species of bats and mammals. The differentiation of spermatogonia is similar to other bats and to Primates, with three main spermatogonia types: A_d, A_p, and B. Meiotic divisions proceed similarly to those of most mammals and spermiogenesis is clearly divided into 12 steps, in the middle of the range of developmental steps for bats (9–16 steps). The process of acrosome formation is similar to that found in Platyrrhinus lineatus, with the acrosome formed by two different types of proacrosomal vesicles. The ultrastructure of the spermatozoon is similar to other bats already described and resembles the typical mammalian sperm model; however, its morphology differs from other mammals such as marsupials and rodents, on account of a simpler spermatozoon head morphology, which indicates a pattern that is more closely related to the sperm cells of humans and other primates. Our data demonstrated that spermatogenesis in C. perspicillata presents great ultrastructural similarities to P. lineatus. This pattern is not surprising, because both species belong to the same family (Phyllostomidae); however, it is observed that C. perspicillata presents some characteristics that are more closely related to phylogenetically distant species, such as Myotis nigricans (Vespertilionidae), which is a fact that deserves attention. J. Morphol. 275:111–123, 2014. © 2013 Wiley Periodicals, Inc.     

Differential Function of the Two Atg4 Homologues in the Aggrephagy Pathway in Caenorhabditis elegans

The presence of multiple homologues of the same yeast Atg protein endows an additional layer of complexity on the autophagy pathway in higher eukaryotes. The physiological function of the individual genes, however, remains largely unknown. Here we investigated the role of the two Caenorhabditis elegans homologues of the cysteine protease Atg4 in the pathway responsible for degradation of protein aggregates. Loss of atg-4.1 activity causes defective degradation of a variety of protein aggregates, whereas atg-4.2 mutants remove these substrates normally. LGG-1 precursors accumulate in atg-4.1 mutants, but not atg-4.2 mutants. LGG-1 puncta, formation of which depends on lipidation of LGG-1, are present in atg-4.1 and atg-4.2 single mutants, but are completely absent in atg-4.1; atg-4.2 double mutants. In vitro enzymatic analysis revealed that ATG-4.1 processes LGG-1 precursors about 100-fold more efficiently than ATG-4.2. Expression of a mutant form LGG-1, which mimics the processed precursor, rescues the defective autophagic degradation of protein aggregates in atg-4.1 mutants and, to a lesser extent, in atg-4.1; atg-4.2 double mutants. Our study reveals that ATG-4.1 and ATG-4.2 are functionally redundant yet display differential LGG-1 processing and deconjugating activity in the aggrephagy pathway in C. elegans.     

Spermiogenesis in the imbricate alligator lizard,Barisia imbricata (Reptilia,Squamata, Anguidae)

Although the events of spermiogenesis are commonly studied in amniotes, the amount of research available for Squamata is lacking. Many studies have described the morphological characteristics of mature spermatozoa in squamates, but few detail the ultrastructural changes that occur during spermiogenesis. This study's purpose is to gain a better understanding of the subcellular events of spermatid development within the Imbricate Alligator Lizard, Barisia imbricata. The morphological data presented here represent the first complete ultrastructural study of spermiogenesis within the family Anguidae. Samples of testes from four specimens collected on the northwest side of the Nevado de Toluca, México, were prepared using standard techniques for transmission electron microscopy. Many of the ultrastructural changes occurring during spermiogenesis within B. imbricata are similar to that of other squamates (i.e., early acrosome formation, chromatin condensation, flagella formation, annulus present, and a prominent manchette). However, there are a few unique characteristics within B. imbricata spermatids that to date have not been described during spermiogenesis in other squamates. For example, penetration of the acrosomal granule into the subacrosomal space to form the basal plate of the perforatorium during round spermatid development, the clover‐shaped morphology of the developing nuclear fossa of the flagellum, and the bulbous shape to the perforatorium are all unique to the Imbricate Alligator Lizard. These anatomical character differences may be valuable nontraditional data that along with more traditional matrices (such as DNA sequences and gross morphological data) may help elucidate phylogenetic relationships, which are historically considered controversial within Squamata. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Spermiogenesis and structure of spermatozoa in the oribatid mite,Hafenrefferia gilvipes (C.L. Koch) (Acari,Oribatida)

The process of spermiogenesis and the structure of spermatozoa in the mite, Hafenrefferia gilvipes (Koch) were studied ultrastructurally. Spermiogenesis was divided into six stages. The spermatids at stage 1 have the usual structure. At stage 2 the structure of the mitochondria and their distribution in the spermatid start to change, leading to the formation of specific mitochondrial derivatives which are subsequently incorporated into the nucleus of the spermatozoon. Parallel to the transformation of mitochondria occurs a reorganization of the nuclear material. The fully formed spermatozoon has a tadpole-like shape, with the cell nucleus located in the distended part of the cell, and containing mitochondrial derivatives in its karyoplasm. Acrosome, flagellum and centrioles are absent. The participation of peripherally distributed microtubules, present in spermatids at stages 4 to 6, in the shaping of the spermatozoon has been suggested.     

埃及尼罗鲶鱼精子形成及精子超微结构(英文)

用透射电镜观察了埃及尼罗鲶鱼(Chrysichthys auratus)精子形成及精子超微结构。精子形成过程除了具有鱼类精子形成的共同特征外,还具有一些特点由于细胞核没有转动,中心粒复合体和鞭毛的起始部分位于细胞核的后端,并与核垂直;精子细胞变态过程中未产生袖套腔;基板未跨越基体的基部;基足将基体固定于细胞核;中段和鞭毛的基部具大量囊泡。成熟精子头部呈长锥状,没有顶体;中段长并含大量囊泡,向后延伸并包围鞭毛的基部;鞭毛细长,无侧鳍;线粒体位于核的后端附近,并包围轴丝;轴丝具典型的“9 2”模式。总之,埃及尼罗鲶鱼的精子形成有别于硬骨鱼类的常见的精子形成类型——I型和II型,可以称为III型。     

A Novel CaM Kinase II Pathway Controls the Location of Neuropeptide Release from Caenorhabditis elegans Motor Neurons

Neurons release neuropeptides via the regulated exocytosis of dense core vesicles (DCVs) to evoke or modulate behaviors. We found that Caenorhabditis elegans motor neurons send most of their DCVs to axons, leaving very few in the cell somas. How neurons maintain this skewed distribution and the extent to which it can be altered to control DCV numbers in axons or to drive release from somas for different behavioral impacts is unknown. Using a forward genetic screen, we identified loss-of-function mutations in UNC-43 (CaM kinase II) that reduce axonal DCV levels by ∼90% and cell soma/dendrite DCV levels by ∼80%, leaving small synaptic vesicles largely unaffected. Blocking regulated secretion in unc-43 mutants restored near wild-type axonal levels of DCVs. Time-lapse video microscopy showed no role for CaM kinase II in the transport of DCVs from cell somas to axons. In vivo secretion assays revealed that much of the missing neuropeptide in unc-43 mutants is secreted via a regulated secretory pathway requiring UNC-31 (CAPS) and UNC-18 (nSec1). DCV cargo levels in unc-43 mutants are similarly low in cell somas and the axon initial segment, indicating that the secretion occurs prior to axonal transport. Genetic pathway analysis suggests that abnormal neuropeptide function contributes to the sluggish basal locomotion rate of unc-43 mutants. These results reveal a novel pathway controlling the location of DCV exocytosis and describe a major new function for CaM kinase II.     

Ultrastructure of spermatogenesis and mature spermatozoa in the flatworm Prosthiostomum siphunculus (Polycladida,Cotylea)

This is the first study investigating spermatogenesis and spermatozoan ultrastructure in the polyclad flatworm Prosthiostomum siphunculus. The testes are numerous and scattered as follicles ventrally between the digestive ramifications. Each follicle contains the different stages of sperm differentiation. Spermatocytes and spermatids derive from a spermatogonium and the spermatids remain connected by intercellular bridges. Chromatoid bodies are present in the cytoplasm of spermatogonia up to spermatids. During early spermiogenesis, a differentiation zone appears in the distal part of spermatids. A ring of microtubules extends along the entire sperm shaft just beneath the cell membrane. An intercentriolar body is present and gives rise to two axonemes, each with a 9 + “1” micro‐tubular pattern. Development of the spermatid leads to cell elongation and formation of a filiform, mature spermatozoon with two free flagella and with cortical microtubules along the sperm shaft. The flagella exit the sperm shaft at different levels, a finding common for acotyleans, but so far unique for cotylean polyclads. The Golgi complex produces numerous electron‐dense bodies of two types and of different sizes. These bodies are located around a perinuclear row of mitochondria. The elongated nucleus extends almost along the entire sperm body. The nucleus is wide in the proximal part and becomes narrow going towards the distal end. Thread‐like chromatin mixed with electron‐dense intranuclear spindle‐shaped bodies are present throughout nucleus. The general sperm ultrastructure, the presence of intranuclear bodies and a second type of cytoplasmic electron‐dense bodies may provide characters useful for phylogenetic analysis.