Early Stages Of Oogenesis Research Articles

Silencing of the 20S proteasomal subunit-α6 triggers full oogenesis arrest and increased mRNA levels of the selective autophagy adaptor protein p62/SQSTM1 in the ovary of the vector Rhodnius prolixus.

The high reproductive rates of insects contribute significantly to their ability to act as vectors of a variety of vector-borne diseases. Therefore, it is strategically critical to find molecular targets with biotechnological potential through the functional study of genes essential for insect reproduction. The ubiquitin-proteasome system is a vital degradative pathway that contributes to the maintenance of regular eukaryotic cell proteostasis. This mechanism involves the action of enzymes to covalently link ubiquitin to proteins that are meant to be delivered to the 26S proteasome and broken down. The 26S proteasome is a large protease complex (including the 20S and 19S subcomplexes) that binds, deubiquitylates, unfolds, and degrades its substrates. Here, we used bioinformatics to identify the genes that encode the seven α and β subunits of the 20S proteasome in the genome of R. prolixus and learned that those transcripts are accumulated into mature oocytes. To access proteasome function during oogenesis, we conducted RNAi functional tests employing one of the 20S proteasome subunits (Prosα6) as a tool to suppress 20S proteasomal activity. We found that Prosα6 silencing resulted in no changes in TAG buildup in the fat body and unaffected availability of yolk proteins in the hemolymph of vitellogenic females. Despite this, the silencing of Prosα6 culminated in the impairment of oocyte maturation at the early stages of oogenesis. Overall, we discovered that proteasome activity is especially important for the signals that initiate oogenesis in R. prolixus and discuss in what manner further investigations on the regulation of proteasome assembly and activity might contribute to the unraveling of oogenesis molecular mechanisms and oocyte maturation in this vector.

Epithelial morphogenesis in the Drosophila egg chamber requires Parvin and ILK.

Integrins are the major family of transmembrane proteins that mediate cell-matrix adhesion and have a critical role in epithelial morphogenesis. Integrin function largely depends on the indirect connection of the integrin cytoplasmic tail to the actin cytoskeleton through an intracellular protein network, the integrin adhesome. What is currently unknown is the role of individual integrin adhesome components in epithelia dynamic reorganization. Drosophila egg chamber consists of the oocyte encircled by a monolayer of somatic follicle epithelial cells that undergo specific cell shape changes. Egg chamber morphogenesis depends on a developmental array of cell-cell and cell-matrix signalling events. Recent elegant work on the role of integrins in the Drosophila egg chamber has indicated their essential role in the early stages of oogenesis when the pre-follicle cells assemble into the follicle epithelium. Here, we have focused on the functional requirement of two key integrin adhesome components, Parvin and Integrin-Linked Kinase (ILK). Both proteins are expressed in the developing ovary from pupae to the adult stage and display enriched expression in terminal filament and stalk cells, while their genetic removal from early germaria results in severe disruption of the subsequent oogenesis, leading to female sterility. Combining genetic mosaic analysis of available null alleles for both Parvin and Ilk with conditional rescue utilizing the UAS/Gal4 system, we found that Parvin and ILK are required in pre-follicle cells for germline cyst encapsulation and stalk cell morphogenesis. Collectively, we have uncovered novel developmental functions for both Parvin and ILK, which closely synergize with integrins in epithelia.

Diversity of Balbiani body formation in internally and externally fertilizing representatives of Osteoglossiformes (Teleostei: Osteoglossomorpha).

During the early stages of oogenesis, the Balbiani body is formed in the primary oocytes. It consists of the Golgi apparatus, endoplasmic reticulum (ER), and numerous mitochondria aggregated with germ plasm, but its form may differ among animals. Hypothetically, during oogenesis oocytes become adapted to future development in two different environments depending on internal or external fertilization. We aimed to investigate, using light and transmission electron microscopy, the development of the Balbiani body during oogenesis in representatives of Osteoglossiformes, one of the most basal Teleostei groups. We analyzed the structure of oogonia and primary oocytes in the internally fertilizing butterflyfish Pantodon buchholzi and the externally fertilizing Osteoglossum bicirrhosum and Arapaima gigas to compare formation of the Balbiani body in relation to modes of fertilization. We demonstrated that the presence of the germ plasm as well as the fusion and fission of mitochondria are the conserved features of the Bb. However, each species exhibited also some peculiar features, including the presence of three types of ooplasm with different electron density and mitochondria-associated membranes in P. buchholzi; annulate lamellae, complexes of the Golgi apparatus, ER network, and lysosome-like bodies in O. bicirrhosum; as well as karmellae and whorls formed by the lamellae of the ER in A. gigas. Moreover, the form of the germ plasm observed in close contact with mitochondria differed between osteoglossiforms, with a "net-like" structure in P. buchholzi, the presence of numerous strings in O. bicirrhosum, and irregular accumulations in A. gigas. These unique features indicate that the extreme diversity of gamete structure observed so far only in the spermatozoa of osteoglossiforms is also characteristic for oocyte development in these basal teleosts. Possible reason of this variability is a period of about 150 million years of independent evolution of the lineages.

Cloning, pattern of gonadal soma-derived factor mRNA in the orange-spotted grouper, Epinephelus coioides

The orange-spotted grouper (Epinephelus coioides), as a protogynous hermaphrodite, provides a novel model for understanding the mechanisms of sex determination and differentiation in teleosts. Gonadal soma-derived factor (gsdf), involved in early germ cell development, is a new member of the transforming growth factor-beta (TGF-β) superfamily. In this study, a 2364-bp long gsdf gene was cloned from E. coioides and there was further analysis of its tissue distribution and patterns of gene expression during the female phase, bisexual gonads stage and testis stage. The cellular localization of Gsdf during the early sexual differentiation stage was also analyzed. In addition, the expression levels of gsdf, dmrt1, foxl2 and cyp19a1a in gonads during the early sexual differentiation stage were analyzed by real-time PCR. Tissue distribution analysis showed that gsdf was only expressed in the testis and ovary. The gene expression showed that gsdf was expressed significantly higher in testes and bisexual gonads compared to ovaries. The early-stage ovary showed a significant fluorescence signal in the supporting cells surrounding gonium and oogonium, but not pre-vitellogenic oocytes, indicating that gsdf expression was restricted to morphologically undifferentiated supporting cells in the early stages of oogenesis. During the early sexual differentiation stage, Real-time PCR showed that both the expression levels of gsdf and dmrt1 were progressively increased and the expression levels of foxl2 and cyp19a1a were growing slowly. These results suggest that gsdf could have roles in the early sexual differentiation in hermaphroditic, E. coioides.

RNA degradation is required for the germ-cell to maternal transition in Drosophila

In sexually reproducing animals, the oocyte contributes a large supply of RNAs that are essential to launch development upon fertilization. The mechanisms that regulate the composition of the maternal RNA contribution during oogenesis are unclear. Here, we show that a subset of RNAs expressed during the early stages of oogenesis is subjected to regulated degradation during oocyte specification. Failure to remove these RNAs results in oocyte dysfunction and death. We identify the RNA-degrading Super Killer complex and No-Go Decay factor Pelota as key regulators of oogenesis via targeted degradation of specific RNAs expressed in undifferentiated germ cells. These regulators target RNAs enriched for cytidine sequences that are bound by the polypyrimidine tract binding protein Half pint. Thus, RNA degradation helps orchestrate a germ cell-to-maternal transition that gives rise to the maternal contribution to the zygote.

Optimal RNA binding by Egalitarian, a Dynein cargo adaptor, is critical for maintaining oocyte fate in Drosophila

ABSTRACT The Dynein motor is responsible for the localization of numerous mRNAs within Drosophila oocytes and embryos. The RNA binding protein, Egalitarian (Egl), is thought to link these various RNA cargoes with Dynein. Although numerous studies have shown that Egl is able to specifically associate with these RNAs, the nature of these interactions has remained elusive. Egl contains a central RNA binding domain that shares limited homology with an exonuclease, yet Egl binds to RNA without degrading it. Mutations have been identified within Egl that disrupt its association with its protein interaction partners, BicaudalD (BicD) and Dynein light chain (Dlc), but no mutants have been described that are specifically defective for RNA binding. In this report, we identified a series of positively charged residues within Egl that are required for RNA binding. Using corresponding RNA binding mutants, we demonstrate that specific RNA cargoes are more reliant on maximal Egl RNA biding activity for their correct localization in comparison to others. We also demonstrate that specification and maintenance of oocyte fate requires maximal Egl RNA binding activity. Even a subtle reduction in Egl’s RNA binding activity completely disrupts this process. Our results show that efficient RNA localization at the earliest stages of oogenesis is required for specification of the oocyte and restriction of meiosis to a single cell.

Effect of mitophagy in oocytes and granulosa cells on oocyte quality†.

Mitophagy is the process by which cells selectively remove supernumerary or damaged mitochondria through autophagy, and is crucial for mitochondrial homeostasis and cell survival. Mitochondria play vital roles in determining the developmental competence of oocytes. During the early stages of oogenesis, aberrant mitochondria can be removed by mitophagy. After oocyte formation, mitophagy is not actively initiated to clear damaged mitochondria despite the presence of mitophagy regulators in oocytes, which leads to the transmission of dysfunctional mitochondria from the oocyte to the embryo. However, granulosa cells around oocytes can improve mitochondrial function through mitophagy, thereby improving oocyte developmental capacity. Furthermore, this review discusses recent work on the substances and environmental conditions that affect mitophagy in oocytes and granulosa cells, thus providing new directions for improving oocyte quality during assisted reproductive technology treatment.

Spatiotemporal expression of activin receptor-like kinase-5 and bone morphogenetic protein receptor type II in the ovary of shortfinned eel, Anguilla australis.

In the eel ovary, the expression of growth differentiation factor-9 (Gdf9) appears to be largely confined to the germ cell in early stages of oogenesis. However, both the target tissue and the function of Gdf9 in fish remain unknown. This study aimed to describe the abundance and localization of activin receptor-like kinase-5 (Alk5) and bone morphogenetic protein receptor type II (Bmpr2), which together mediate the Gdf9 signal, in the ovary of a basal teleost, the shortfinned eel, Anguilla australis, during early folliculogenesis. The cDNA encoding eel alk5 and bmpr2 genes were cloned, characterized and the transcript abundances of these receptors quantified by quantitative real-time PCR. Ovarian transcript abundance for both receptors, along with that of gdf9 and of its paralogue bmp15, increased from the previtellogenic to early vitellogenic stage. Localization of receptor mRNAs by in situ hybridization revealed that these receptors are located in the somatic cells surrounding the oocyte. Furthermore, tissue distribution analysis showed that the expression of alk5 and bmpr2 were highest in ovary and thyroid, respectively. Unexpectedly, however, bmpr2 mRNA levels were lower in the ovary than in any of the other 17 tissues examined, and indeed, lower than ovarian gdf9 transcript abundance. These findings, together with the ovarian expression pattern of Gdf9, suggest that Gdf9, and conceivably, Bmp15, from the oocyte can signal through receptors that are located on the somatic cells surrounding the oocyte; this, in turn, facilitates elucidation of the function of these growth factors during oogenesis in teleost fish.

A putative stem-loop structure in Drosophila crumbs is required for mRNA localisation in epithelia and germline cells.

Crumbs (Crb) is an evolutionarily conserved transmembrane protein localised to the apical membrane of epithelial cells. Loss or mislocalisation of Crb is often associated with disruption of apicobasal cell polarity. crb mRNA is also apically enriched in epithelial cells, and, as shown here, accumulates in the oocyte of developing egg chambers. We narrowed down the localisation element (LE) of crb mRNA to 47 nucleotides, which form a putative stem-loop structure that may be recognised by Egalitarian (Egl). Mutations in conserved nucleotides abrogate apical transport. crb mRNA enrichment in the oocyte is affected in egl mutant egg chambers. A CRISPR-based genomic deletion of the crb locus that includes the LE disrupts asymmetric crb mRNA localisation in epithelia and prevents its accumulation in the oocyte during early stages of oogenesis, but does not affect Crb protein localisation in embryonic and follicular epithelia. However, flies lacking the LE show ectopic Crb protein expression in the nurse cells. These data suggest an additional role for the Drosophila 3'-UTR in regulating translation in a tissue-specific manner.This article has an associated First Person interview with the first author of the paper.

Reproductive biology of the Neotropical catfish Iheringichthys labrosus (Siluriformes: Pimelodidae), with anatomical and morphometric analysis of gonadal tissues

The reproduction of Iheringichthys labrosus (Lütken, 1874) from the Turvo River, Brazil, was studied using anatomical, biometric, histological, and ultrastructural techniques. Between April 2014 and March 2015, a total of 278 males and 512 females were captured bimonthly. The testes of Iheringichthys labrosus are fringed and possess a cranial spermatogenic region and an exclusively secretory caudal region. Histologically, the cranial region is composed of seminiferous tubules with spermatogenesis being completed in cysts. The spermatozoa are of the primitive type with a spherical head and have a rudimentary intermediate piece and a long tail with an axonemic arrangement of 9 + 2. The caudal region does not form an individualized gland, and cells in this testis area have characteristics of protein secretion. A variable density electron-dense secretion accumulates in the cisternae of the rough endoplasmic reticulum in the lumen of the seminiferous tubules and in the testicular ducts during maturation. The cortical alveoli are discontinuous, and the zona pellucida consists of three layers crossed by pore canals, and the follicular cells are squamous in the early stages of oogenesis and cuboidal in advanced stages. The gonadosomatic index was associated with the maturation of the gonads while the condition factor indicated that the fish feed less and utilize adipose reserves during the reproductive period. Males and females reproductively functional throughout the year with spawning being partial or multiple, similar to that reported in studies of the species in lentic environments.

Identification and characterization of POU class V family genes in Japanese red bellied newt, Cynops pyrrhogaster.

Mammalian Pou5f1 encodes the POU family class V (POU-V) transcription factor which is essential for the pluripotency of embryonic cells and germ cells. In vertebrates, various POU-V family genes have been identified and classified into the POU5F1 family or its paralogous POU5F3 family. In this study, we cloned two cDNAs named CpPou5f1 and CpPou5f3, which encode POU-V family proteins of the Japanese red bellied newt Cynops pyrrhogaster. In the predicted amino acid sequence encoded by CpPou5f1, the typical MAGH sequence at the N-terminus and deletion of arginine at the fifth position of POU-homeodomain were recognized, but not in the sequence encoded by CpPou5f3. Phylogenetic analysis using Clustal Omega software indicated that CpPou5f1 and CpPou5f3 are classified into the clade of the POU5F1 and POU5F3 families, respectively. In a real-time polymerase chain reaction (RT-PCR) analysis, the marked gene expression of CpPou5f1 was observed during oogenesis and early development up to the tail-bud stage, whereas weak gene expression of CpPou5f3 was detected only in the early stages of oogenesis and gastrula. In adult organs, CpPou5f1 was expressed only in the ovary, while gene expression of CpPou5f3 was recognized in various organs. A regeneration experiment using larval forelimb revealed that transient gene expression of CpPou5f1 occurred at the time of wound healing, followed by gene activation of CpPou5f3 during the period of blastema formation. These results suggest that CpPou5f1 and CpPou5f3 might play different roles in embryogenesis and limb regeneration.

Dynamics of the spatial orientation of the pericentromeric heterochromatin regions in the polytene chromosomes of ovarian nurse cells in the Drosophila melanogaster (Diptera: Drosophilidae) oogenesis

The spatial organization of polytene chromosomes and the association of their pericentromeric regions in the Drosophila melanogaster nurse cells during oogenesis have been examined by 3D-immunofluorescence microscopy. All nurse cell chromosomes are shown to contact the nuclear membrane with their pericentromeric regions, and the X chromosome additionally with its telomeric region. Three morphological types of the associations of the nurse cell chromosome pericentromeric regions in the nuclear space are observed: (1) the pericentromeric regions of chromosomes 2, 3, and 4 contact the nuclear membrane at one pole of the nucleus, while the pericentromeric region of the X chromosome does so at the other pole (morphotype I); (2) the pericentromeric regions of chromosomes X, 2, and 3 are separated from each other in the nuclear space and contact the nuclear membrane (morphotype II); and (3) the pericentromeric region of chromosome 2 contacts the nuclear membrane at one pole of the nucleus, while the pericentromeric regions of chromosomes X, 3, and 4 contact the membrane at the other pole (morphotype III). The author, therefore, proposes that the nurse cell nuclei with morphotype I are prevalent at the early stages of oogenesis, while the nurse cell nuclei with morphotype III are most abundant at the late stages. The dynamics of associations of the pericentromeric chromosome regions in the nuclear space of D. melanogaster ovarian nurse cells in the oogenesis demonstrated that there may be some functional relationship between the 3-D organization of the nurse cell chromosomes and the organization of the nucleolus.

Receptor-interacting Ser/Thr kinase 1 (RIPK1) and myosin IIA–dependent ceramidosomes form membrane pores that mediate blebbing and necroptosis

Formation of membrane pores/channels regulates various cellular processes, such as necroptosis or stem cell niche signaling. However, the roles of membrane lipids in the formation of pores and their biological functions are largely unknown. Here, using the cellular stress model evoked by the sphingolipid analog drug FTY720, we show that formation of ceramide-enriched membrane pores, referred to here as ceramidosomes, is initiated by a receptor-interacting Ser/Thr kinase 1 (RIPK1)-ceramide complex transported to the plasma membrane by nonmuscle myosin IIA-dependent trafficking in human lung cancer cells. Molecular modeling/simulation coupled with site-directed mutagenesis revealed that Asp147 or Asn169 of RIPK1 are key for ceramide binding and that Arg258 or Leu293 residues are involved in the myosin IIA interaction, leading to ceramidosome formation and necroptosis. Moreover, generation of ceramidosomes independently of any external drug/stress stimuli was also detected in the plasma membrane of germ line stem cells in ovaries during the early stages of oogenesis in Drosophila melanogaster Inhibition of ceramidosome formation via myosin IIA silencing limited germ line stem cell signaling and abrogated oogenesis. In conclusion, our findings indicate that the RIPK1-ceramide complex forms large membrane pores we named ceramidosomes. They further suggest that, in addition to their roles in stress-mediated necroptosis, these ceramide-enriched pores also regulate membrane integrity and signaling and might also play a role in D. melanogaster ovary development.

Subcellular localization and Egl-mediated transport of telomeric retrotransposon HeT-A ribonucleoprotein particles in the Drosophila germline and early embryogenesis.

The study of the telomeric complex in oogenesis and early development is important for understanding the mechanisms which maintain genome integrity. Telomeric transcripts are the key components of the telomeric complex and are essential for regulation of telomere function. We study the biogenesis of transcripts generated by the major Drosophila telomere repeat HeT-A in oogenesis and early development with disrupted telomeric repeat silencing. In wild type ovaries, HeT-A expression is downregulated by the Piwi-interacting RNAs (piRNAs). By repressing piRNA pathway, we show that overexpressed HeT-A transcripts interact with their product, RNA-binding protein Gag-HeT-A, forming ribonucleoprotein particles (RNPs) during oogenesis and early embryonic development. Moreover, during early stages of oogenesis, in the nuclei of dividing cystoblasts, HeT-A RNP form spherical structures, which supposedly represent the retrotransposition complexes participating in telomere elongation. During the later stages of oogenesis, abundant HeT-A RNP are detected in the cytoplasm and nuclei of the nurse cells, as well as in the cytoplasm of the oocyte. Further on, we demonstrate that HeT-A products co-localize with the transporter protein Egalitarian (Egl) both in wild type ovaries and upon piRNA loss. This finding suggests a role of Egl in the transportation of the HeT-A RNP to the oocyte using a dynein motor. Following germline piRNA depletion, abundant maternal HeT-A RNP interacts with Egl resulting in ectopic accumulation of Egl close to the centrosomes during the syncytial stage of embryogenesis. Given the essential role of Egl in the proper localization of numerous patterning mRNAs, we suggest that its abnormal localization likely leads to impaired embryonic axis specification typical for piRNA pathway mutants.

Pleiotropic Functions of the Chromodomain-Containing Protein Hat-trick During Oogenesis in Drosophila melanogaster.

Chromatin-remodeling proteins have a profound role in the transcriptional regulation of gene expression during development. Here, we have shown that the chromodomain-containing protein Hat-trick is predominantly expressed within the oocyte nucleus, specifically within the heterochromatinized karyosome, and that a mild expression is observed in follicle cells. Colocalization of Hat-trick with Heterochromatin Protein 1 and synaptonemal complex component C(3)G along with the diffused karyosome after hat-trick downregulation shows the role of this protein in heterochromatin clustering and karyosome maintenance. Germline mosaic analysis reveals that hat-trick is required for maintaining the dorso-ventral patterning of eggs by regulating the expression of Gurken. The increased incidence of double-strand breaks (DSBs), delayed DSB repair, defects in karyosome formation, altered Vasa mobility, and, consequently, misexpression and altered localization of Gurken in hat-trick mutant egg chambers clearly suggest a putative involvement of Hat-trick in the early stages of oogenesis. In addition, based on phenotypic observations in hat-trick mutant egg chambers, we speculate a substantial role of hat-trick in cystoblast proliferation, oocyte determination, nurse cell endoreplication, germ cell positioning, cyst encapsulation, and nurse cell migration. Our results demonstrate that hat-trick has profound pleiotropic functions during oogenesis in Drosophila melanogaster.

Identification and expression analysis of microRNAs in medaka gonads

Gonad development is a highly regulated, coordinated biological process and increasing evidences have indicated that microRNA (miRNA) may be involved in this dynamic program. Medaka (Oryzias latipes) is a good model for reproductive research as it has distinct sex determining genes, however, research in gonadal miRNAs is lacked. In this study, two small RNA libraries from the ovaries and testes were constructed and sequenced. A total of 285 conserved and 388 novel miRNAs were obtained, among which 142 mature miRNAs were significantly (> two-fold change) up or down regulated in the testis compared to the ovary. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis showed that miR-430c, miR-26a and miR-202-5p were expressed in a gonad-specific or sex-biased pattern. Fluorescence in situ hybridization (FISH) indicated that miR-202-5p was present throughout spermatogenesis and was only detected at the early stages of oogenesis, this sex biased expression pattern suggested that miR-202-5p might be a crucial candidate in male differentiation and development. Our study provides the repertoire, a comprehensive annotation of miRNAs from gonads and a reference for functional studies of miRNAs in medaka.

Micromorphology of ovaries and oogenesis in Grania postclitellochaeta (Clitellata: Enchytraeidae)

The genus Grania comprises over 70 species of exclusively marine clitellate annelids belonging to the family Enchytraeidae. Morphologically, this genus is well separated from other enchytraeids, with thick cuticles, anterior segments I–IV fused into a “head”, chaetal bundles consisting only of one stout chaeta, and reduction of circular musculature. The aim of the present study is to describe the ovary organization and the course of oogenesis in Grania postclitellochaeta, and to compare it with other known systems of ovary organization and oogenesis in clitellate annelids, especially in enchytraeids. Generally, oogenesis in G. postclitellochaeta can be divided into two phases: (i) early stages of oogenesis, occurring within the paired ovaries − each ovary is similar to a bunch of grapes, where each ‘lobe’ is a germ-line cyst enveloped by flat somatic cells, and (ii) oogenesis proper, which takes place within the body lumen where each growing oocyte is accompanied by its own group of nurse cells. Germ cells are interconnected by cytoplasmic channels (intercellular bridges, ring canals) and form syncytial cysts. As in other clitellate annelids, the cyst center contains a common cytoplasm (cytophore) to which each cell is connected by one ring canal only. Initially, within the ovary, all interconnected cells develop synchronously and are morphologically similar. At the time when the cysts detach from the ovary, one of the interconnected cells begins to gather nutrients, grows and becomes an oocyte, whereas the rest of the cells (nurse cells) do not continue meiosis and instead seem to provide the oocyte with macromolecules and cell organelles. Analysis of serial sections reveals that cysts are always composed of 16 cells − one oocyte and fifteen nurse cells. A comparative analysis showed that almost all features of oogenesis in G. postclitellochaeta are similar to that in other representatives of Enchytraeidae (mainly Enchytraeus albidus), suggesting evolutionary conservation of the process across this family.

Notch signalling mediates reproductive constraint in the adult worker honeybee.

The hallmark of eusociality is the reproductive division of labour, in which one female caste reproduces, while reproduction is constrained in the subordinate caste. In adult worker honeybees (Apis mellifera) reproductive constraint is conditional: in the absence of the queen and brood, adult worker honeybees activate their ovaries and lay haploid male eggs. Here, we demonstrate that chemical inhibition of Notch signalling can overcome the repressive effect of queen pheromone and promote ovary activity in adult worker honeybees. We show that Notch signalling acts on the earliest stages of oogenesis and that the removal of the queen corresponds with a loss of Notch protein in the germarium. We conclude that the ancient and pleiotropic Notch signalling pathway has been co-opted into constraining reproduction in worker honeybees and we provide the first molecular mechanism directly linking ovary activity in adult worker bees with the presence of the queen.

Clathrin heavy chain plays multiple roles in polarizing the Drosophila oocyte downstream of Bic-D

Bicaudal-D (Bic-D), Egalitarian (Egl), microtubules and their motors form a transport machinery that localizes a remarkable diversity of mRNAs to specific cellular regions during oogenesis and embryogenesis. Bic-D family proteins also promote dynein-dependent transport of Golgi vesicles, lipid droplets, synaptic vesicles and nuclei. However, the transport of these different cargoes is still poorly understood. We searched for novel proteins that either mediate Bic-D-dependent transport processes or are transported by them. Clathrin heavy chain (Chc) co-immunopurifies with Bic-D in embryos and ovaries, and a fraction of Chc colocalizes with Bic-D. Both proteins control posterior patterning of the Drosophila oocyte and endocytosis. Although the role of Chc in endocytosis is well established, our results show that Bic-D is also needed for the elevated endocytic activity at the posterior of the oocyte. Apart from affecting endocytosis indirectly by its role in osk mRNA localization, Bic-D is also required to transport Chc mRNA into the oocyte and for transport and proper localization of Chc protein to the oocyte cortex, pointing to an additional, more direct role of Bic-D in the endocytic pathway. Furthermore, similar to Bic-D, Chc also contributes to proper localization of osk mRNA and to oocyte growth. However, in contrast to other endocytic components and factors of the endocytic recycling pathway, such as Rabenosyn-5 (Rbsn-5) and Rab11, Chc is needed during early stages of oogenesis (from stage 6 onwards) to localize osk mRNA correctly. Moreover, we also uncovered a novel, presumably endocytosis-independent, role of Chc in the establishment of microtubule polarity in stage 6 oocytes.

Novel Strain of Spiroplasma Found in Flower Bugs of the Genus Orius (Hemiptera: Anthocoridae): Transovarial Transmission, Coexistence with Wolbachia and Varied Population Density

Spiroplasma, a group of small, wall-less, helical, and motile bacteria belonging to the Mollicutes, contains species with diverse life histories. To date, all the Spiroplasma strains that are known to be transmitted vertically in arthropod lineages belong to either the Spiroplasma ixodetis group or the Spiroplasma poulsonii group. Here, we found that a unique strain of Spiroplasma vertically transmitted in predatory flower bugs of the genus Orius belongs to the Spiroplasma insolitum group, which is a group of bacteria phylogenetically closely related to S. insolitum derived from the tickseed sunflower, Bidens sp. (Asterales: Asteraceae). The infection frequencies in natural populations were16.0% in Orius sauteri (n = 75), 40.5% in Orius nagaii (n = 37), and 8.0% in Orius minutus (n = 87). Orius strigicollis was not infected with Spiroplasma (n = 147). In the early stage of oogenesis (i.e., within the germarium), a large number of bacteria with the typical morphology of Spiroplasma existed, keeping a distance from Wolbachia bacteria. The Spiroplasma population seemed to increase during host development but Wolbachia population did not.