Late Oogenesis Research Articles

The bHLH-PAS transcriptional complex Sim:Tgo plays active roles in late oogenesis to promote follicle maturation and ovulation.

Across species, ovulation is a process induced by a myriad of signaling cascades that ultimately leads to the release of encapsulated oocytes from follicles. Follicles first need to mature and gain ovulatory competency before ovulation; however, the signaling pathways regulating follicle maturation are incompletely understood in Drosophila and other species. Our previous work has shown that the bHLH-PAS transcription factor Single-minded (Sim) plays important roles in follicle maturation downstream of the nuclear receptor Ftz-f1 in Drosophila. Here, we demonstrate that Tango (Tgo), another bHLH-PAS protein, acts as a co-factor of Sim to promote follicle cell differentiation from stages 10 to 12. In addition, we discover that re-upregulation of Sim in stage-14 follicle cells is also essential to promote ovulatory competency by upregulating octopamine receptor in mushroom body (OAMB), matrix metalloproteinase 2 (Mmp2) and NADPH oxidase (NOX), either independently of or in conjunction with the zinc-finger protein Hindsight (Hnt). All these factors are crucial for successful ovulation. Together, our work indicates that the transcriptional complex Sim:Tgo plays multiple roles in late-stage follicle cells to promote follicle maturation and ovulation.

Distinct Wolbachia localization patterns in oocytes of diverse host species reveal multiple strategies of maternal transmission.

A broad array of endosymbionts radiate through host populations via vertical transmission, yet much remains unknown concerning the cellular basis, diversity, and routes underlying this transmission strategy. Here, we address these issues, by examining the cellular distributions of Wolbachia strains that diverged up to 50 million years ago in the oocytes of 18 divergent Drosophila species. This analysis revealed 3 Wolbachia distribution patterns: (1) a tight clustering at the posterior pole plasm (the site of germline formation); (2) a concentration at the posterior pole plasm, but with a significant bacteria population distributed throughout the oocyte; and (3) a distribution throughout the oocyte, with none or very few located at the posterior pole plasm. Examination of this latter class indicates Wolbachia accesses the posterior pole plasm during the interval between late oogenesis and the blastoderm formation. We also find that 1 Wolbachia strain in this class concentrates in the posterior somatic follicle cells that encompass the pole plasm of the developing oocyte. In contrast, strains in which Wolbachia concentrate at the posterior pole plasm generally exhibit no or few Wolbachia in the follicle cells associated with the pole plasm. Taken together, these studies suggest that for some Drosophila species, Wolbachia invade the germline from neighboring somatic follicle cells. Phylogenomic analysis indicates that closely related Wolbachia strains tend to exhibit similar patterns of posterior localization, suggesting that specific localization strategies are a function of Wolbachia-associated factors. Previous studies revealed that endosymbionts rely on 1 of 2 distinct routes of vertical transmission: continuous maintenance in the germline (germline-to-germline) or a more circuitous route via the soma (germline-to-soma-to-germline). Here, we provide compelling evidence that Wolbachia strains infecting Drosophila species maintain the diverse arrays of cellular mechanisms necessary for both of these distinct transmission routes. This characteristic may account for its ability to infect and spread globally through a vast range of host insect species.

Selective Destabilization of Transcripts by mRNA Decapping Regulates Oocyte Maturation and Innate Immunity Gene Expression during Ageing in C. elegans.

Removal of the 5' cap structure of RNAs (termed decapping) is a pivotal event in the life of cytoplasmic mRNAs mainly catalyzed by a conserved holoenzyme, composed of the catalytic subunit DCP2 and its essential cofactor DCP1. While decapping was initially considered merely a step in the general 5'-3' mRNA decay, recent data suggest a great degree of selectivity that plays an active role in the post-transcriptional control of gene expression, and regulates multiple biological functions. Studies in Caenorhabditis elegans have shown that old age is accompanied by the accumulation of decapping factors in cytoplasmic RNA granules, and loss of decapping activity shortens the lifespan. However, the link between decapping and ageing remains elusive. Here, we present a comparative microarray study that was aimed to uncover the differences in the transcriptome of mid-aged dcap-1/DCP1 mutant and wild-type nematodes. Our data indicate that DCAP-1 mediates the silencing of spermatogenic genes during late oogenesis, and suppresses the aberrant uprise of immunity gene expression during ageing. The latter is achieved by destabilizing the mRNA that encodes the transcription factor PQM-1 and impairing its nuclear translocation. Failure to exert decapping-mediated control on PQM-1 has a negative impact on the lifespan, but mitigates the toxic effects of polyglutamine expression that are involved in human disease.

Tracking Follicle Cell Development.

Somatic follicle cells are critical support cells for Drosophila oogenesis, as they provide signals and molecules needed to produce a mature egg. Throughout this process, the follicle cells differentiate into multiple subpopulations and transition between three different cell cycle programs to support nurse cell and oocyte development. The follicle cells are mitotic in early egg chamber development, as they cover the germline cyst. In mid-oogenesis, follicle cells switch from mitosis to endocycling, increasing their ploidy from 2C to 16C. Finally, in late oogenesis, cells transition from endocycling to gene amplification, increasing the copy number of a small subset of genes, including the genes encoding proteins required for egg maturation. In order to explore the genetic regulation of these cell cycle switches and follicle cell development and specification, clonal analysis and the GAL4/UAS system are used frequently to reduce or increase expression of genes of interest. These genetic approaches combined with immunohistochemistry and in situ hybridization are powerful tools for characterizing the mechanisms regulating follicle cell development and the mitosis/endocycle and endocycle/gene amplification transitions. This chapter describes the genetic tools available to manipulate gene expression in follicle cells, as well as the methods and reagents that can be utilized to explore gene expression throughout follicle cell development.

A transition phase in late mouse oogenesis impacts DNA methylation of the early embryo

A well-orchestrated program of oocyte growth and differentiation results in a developmentally competent oocyte. In late oogenesis, germinal vesicle oocytes (GVOs) undergo chromatin remodeling accompanied by transcriptional silencing from an NSN (non-surrounded nucleolus) to an SN (surrounded nucleolus) chromatin state. By analyzing different cytoplasmic and nuclear characteristics, our results indicate that murine NSN-GVOs transition via an intermediate stage into SN-GVOs in vivo. Interestingly, this transition can also be observed ex vivo, including most characteristics seen in vivo, which allows to analyze this transition process in more detail. The nuclear rearrangements during the transition are accompanied by changes in DNA methylation and Tet enzyme-catalyzed DNA modifications. Early parthenogenetic embryos, derived from NSN-GVOs, show lower DNA methylation levels than SN-derived embryos. Together, our data suggest that a successful NSN-SN transition in oogenesis including proper DNA methylation remodeling is important for the establishment of a developmentally competent oocyte for the beginning of life.

Comparison of RNA localization during oogenesis within Acipenser ruthenus and Xenopus laevis.

The oocyte is a unique cell, from which develops a complex organism comprising of germ layers, tissues and organs. In some vertebrate species it is known that the asymmetrical localization of biomolecules within the oocyte is what drives the spatial differentiation of the daughter cells required for embryogenesis. This asymmetry is first established to produce an animal-vegetal (A-V) axis which reflects the future specification of the ectoderm, mesoderm, and endoderm layers. Several pathways for localization of vegetal maternal transcripts have already been described using a few animal models. However, there is limited information about transcripts that are localized to the animal pole, even though there is accumulating evidence indicating its active establishment. Here, we performed comparative TOMO-Seq analysis on two holoblastic cleavage models: Xenopus laevis and Acipenser ruthenus oocytes during oogenesis. We found that there were many transcripts that have a temporal preference for the establishment of localization. In both models, we observed vegetal transcript gradients that were established during either the early or late oogenesis stages and transcripts that started their localization during the early stages but became more pronounced during the later stages. We found that some animal gradients were already established during the early stages, however the majority were formed during the later stages of oogenesis. Some of these temporally localized transcripts were conserved between the models, while others were species specific. Additionally, temporal de novo transcription and also degradation of transcripts within the oocyte were observed, pointing to an active remodeling of the maternal RNA pool.

P-531 Influence of tandemly repetitive pericentromeric DNA transcripts inactivation on the formation of membraneless RNP structures at the end of human oocytes maturation

Abstract Study question Does inactivation of tandemly repetitive (TR) pericentromeric DNA transcription affect the size and the number of DDX4-containing RNPs at the end of human oocytes maturation? Summary answer Antisense oligonucleotides (AO) can inactivate the transcription of HS2/HS3. Inactivation leads to disaggregation of the DDX4-based membraneless RNP structures. What is known already Earlier we had described for the first time the transcription of the pericentromeric TR DNA of HS2/HS3 families in late human oogenesis, during the transition of oocytes from the GV stage to the MI stage. Both HS2/HS3 strands were transcribed. The presence of HS2/HS3 transcripts was demonstrated by bioinformatic analysis, qPCR and RNA-DNA-FISH. HS2/HS3 RNAs were found in RNPs which contained DDX5 and DDX4 RNA helicases, located near mitochondria. We suggested that the RNPs could be the site of spatial sequestration of RNA and proteins at the end of human oocytes maturation. Study design, size, duration For studying the inactivation of HS2/HS3 transcription, oocytes were subjected to cytoplasmic microinjection using a micromanipulator. Before the experiment, oocytes were divided into 3 groups (n = 11). Group 1 (control, n = 4) got a dose of saline, group 2 (n = 3) – 8.5 fmol AO, group 3 (n = 4) – 12.75 fmol AO. AO or saline was injected into oocytes, the cells were then placed in a CO2 incubator, and after 48 hours, they were fixed and examined. Participants/materials, setting, methods GV and MI human oocytes are not used for the bank of donor oocytes and were transferred for research. The informed consent of the donors, as well as the permission of the ethical committee of the clinic, was obtained. The distribution of HS2/HS3 transcripts and DDX4 helicase was studied using the immuno-FISH. Fluorescent signals were analyzed using the ImageJ program. Statistical processing of the count data was carried out using GraphPad Prism 9 software. Main results and the role of chance Statistically significant inactivation of TR RNA HS2/HS3 (p = 0.01) in group 3 (AO, 12.75 fmol) was observed. The fluorescence of the HS2/HS3 RNA hybridization signal dropped to 25.68±1.777 as compared to the control group 1 (35.50±2.938). In group 2, this value (34.47 ± 1.984) did not change significantly as compared to the control group. Also, in the 3rd group, there was a tendency (p = 0.16) of an increase in the fluorescence of the antibodies signal against DDX4 up to 26.23 ± 2.223 in comparison with group 1 (19.57 ± 2.496). In group 2, this indicator (21.63 ± 3.095) changed to a lesser extent as compared to the control group. At the same time, in the oocytes of the 3rd group, there was a tendency (p = 0.19) to an increase in the number of individual cytoplasmic foci stained with the antibody against DDX4 up to 76±18.56 as compared with group 1 (44±8.994 signals). In group 2 this value (26±5.508 signals) changed less compared to control group 1. We assume, after the inactivation of HS2/HS3 transcription, DDX4-containing RNPs dissociate since an increase in the number of individual signals of the corresponding DDX4 was revealed. Limitations, reasons for caution The small sample size did not allow us to obtain statistically reliable data on the fluorescence of DDX 4. Data accumulation will be continued in future studies. Wider implications of the findings The data presented confirm the existence of DDX4-based RNPs in human preovulatory oocytes. These membraneless structures are involved in the processes of oocyte maturation. The suggested approach of transcripts inactivation can be used for oocytes without removing zona pellucidae increasing their survival after inactivation. Trial registration number not applicable

Seasonal dynamics, kinetics, and effects of 2-hydroxyestradiol-17β on some steroidogenic enzymes in the ovary of the catfish Heteropneustesfossilis

The synthesis of bioactive steroids is catalyzed by an array of enzymes of diverse properties and actions. In the present study, seasonal dynamics and kinetics of key steroidogenic enzymes, 17α-hydroxylase (Cyp17a), 3β-hydroxysteroid dehydrogenase (Hsd3b), 20α-hydroxysteroid dehydrogenase (Hsd20a) and 20β-hydroxysteroid dehydrogenase (Hsd20b) were investigated in the female catfish Heteropneustes fossilis. Further, the effects of the estrogen metabolite 2-hydroxyestradiol-17β (2-OHE2) and human chorionic gonadotropin (hCG) on activities of the above enzymes, cytochrome P450 aromatase (Cyp19a1), and steroid products including testosterone and cortisol were determined. The enzymes under investigation showed significant seasonal variations across the annual ovarian cycle with low activity in the gonad resting phase. The enzymes Hsd3b and Cyp17a showed high activity during early oogenesis but the activities of Hsd20a and Hsd20b were higher towards late oogenesis in the spawning phase. Hsd3b and Cyp17a elicited high apparent Km values (low substrate affinity) and high apparent Vmax in the vitellogenic phase compared to the postvitellogenic phase. Hsd20a did not elicit any significant differences in the kinetic parameters between the two phases. Hsd20b showed high apparent Km values (low substrate affinity) and high Vmax in the postvitellogenic phase. The incubation of ovarian slices with 2-OHE2 for 24 h increased dose-dependently Hsd3b, Cyp17a, Hsd20a and Hsd20b activities, similar to hCG. The levels of the corresponding C21 steroid products, progesterone (P4), 17α-hydroxyprogesterone (17-OHP4), 17, 20α-dihydroxy-4-pregnen-3-one (17,20α-DP) and 17,20β-dihydoxy-4-pregnen-3-one (17,20β-DP), and cortisol were elevated. However, 2-OHE2 decreased significantly the C19 and C18 steroids, testosterone and E2 levels, and Cyp19a activity. The co-incubation with hCG and 2-OHE2 produced a synergistic effect on the enzyme activities except that of CYP19a. The co-incubation reversed the inhibitory effect of 2-OHE2. The data show that 2-OHE2 exerts a dual role on steroidogenesis, stimulating the C21 pathway and inhibiting the C19–C18 pathway, resulting in the steroidogenic shift.

Shotgun and TMT-Labeled Proteomic Analysis of the Ovarian Proteins of an Insect Vector, Aedes aegypti (Diptera: Culicidae).

Aedes aegypti [Linnaeus in Hasselquist; yellow fever mosquito] transmits several viruses that infect millions of people each year, including Zika, dengue, yellow fever, chikungunya, and West Nile. Pathogen transmission occurs during blood feeding. Only the females blood feed as they require a bloodmeal for oogenesis; in the bloodmeal, holo-transferrin and hemoglobin provide the females with a high iron load. We are interested in the effects of the bloodmeal on the expression of iron-associated proteins in oogenesis. Previous data showed that following digestion of a bloodmeal, ovarian iron concentrations doubles by 72 hr. We have used shotgun proteomics to identify proteins expressed in Ae. aegypti ovaries at two oogenesis developmental stages following blood feeding, and tandem mass tag-labeling proteomics to quantify proteins expressed at one stage following feeding of a controlled iron diet. Our findings provide the first report of mosquito ovarian protein expression in early and late oogenesis. We identify proteins differentially expressed in the two oogenesis development stages. We establish that metal-associated proteins play an important role in Ae. aegypti oogenesis and we identify new candidate proteins that might be involved in mosquito iron metabolism. Finally, this work identified a unique second ferritin light chain subunit, the first reported in any species. The shotgun proteomic data are available via ProteomeXchange with identifier PXD005893, while the tandem mass tag-labeled proteomic data are available with identifier PXD028242.

The IRM cell adhesion molecules Hibris, Kin of irre and Roughest control egg morphology by modulating ovarian muscle contraction in Drosophila

The Irre Cell Recognition Module (IRM) is an evolutionarily conserved group of transmembrane glycoproteins required for cell–cell recognition and adhesion in metazoan development. In Drosophila melanogaster ovaries, four members of this group – Roughest (Rst), Kin of irre (Kirre), Hibris (Hbs) and Sticks and stones (Sns) – play important roles in germ cell encapsulation and muscle sheath organization during early pupal stages, as well as in the progression to late oogenesis in the adult. Females carrying some of the mutant rst alleles are viable but sterile, and previous work from our laboratory had identified defects in the organization of the peritoneal and epithelial muscle sheaths of these mutants that could underlie their sterile phenotype. In this study, besides further characterizing the sterility phenotype associated with rst mutants, we investigated the role of the IRM molecules Rst, Kirre and Hbs in maintaining the functionality of the ovarian muscle sheaths. We found that knocking down any of the three genes in these structures, either individually or in double heterozygous combinations, not only decreases contraction frequency but also irregularly increases contraction amplitude. Furthermore, these alterations can significantly impact the morphology of eggs laid by IRM-depleted females demonstrating a hitherto unknown role of IRM molecules in egg morphogenesis.

Identification of New Regulators of the Oocyte-to-Embryo Transition in Drosophila.

At the oocyte-to-embryo transition the highly differentiated oocyte arrested in meiosis becomes a totipotent embryo capable of embryogenesis. Oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest and the trigger for the oocyte-to-embryo transition) serve as prerequisites for this transition, both events being controlled posttranscriptionally. Recently, we obtained a comprehensive list of proteins whose levels are developmentally regulated during these events via a high-throughput quantitative proteomic analysis of Drosophila melanogaster oocyte maturation and egg activation. We conducted a targeted screen for potential novel regulators of the oocyte-to-embryo transition, selecting 53 candidates from these proteins. We reduced the function of each candidate gene using transposable element insertion alleles and RNAi, and screened for defects in oocyte maturation or early embryogenesis. Deletion of the aquaporin gene CG7777 did not affect female fertility. However, we identified CG5003 and nebu (CG10960) as new regulators of the transition from oocyte to embryo. Mutations in CG5003, which encodes an F-box protein associated with SCF-proteasome degradation function, cause a decrease in female fertility and early embryonic arrest. Mutations in nebu, encoding a putative glucose transporter, result in defects during the early embryonic divisions, as well as a developmental delay and arrest. nebu mutants also exhibit a defect in glycogen accumulation during late oogenesis. Our findings highlight potential previously unknown roles for the ubiquitin protein degradation pathway and sugar transport across membranes during this time, and paint a broader picture of the underlying requirements of the oocyte-to-embryo transition.

Capital Breeding in a Diapausing Copepod: A Transcriptomics Analysis

Capital breeders must balance the energetic requirements of cellular function and the cost of reproduction in organisms. The subarctic copepod Neocalanus flemingeri, a small planktonic crustacean, depends on a short annual phytoplankton bloom to acquire the energy needed to support a non-feeding female adult that enters a period of diapause prior to spawning five to seven months later. After emergence from diapause, the reproductive program takes two-months starting with germline development. The relationships between energy utilization, cellular maintenance and oogenesis were investigated through gene expression profiling. The sequential up- and down-regulation of genes involved in cellular maintenance and metabolic pathways coincided with four developmental groups: diapause, emergence from diapause and early oogenesis, mid to late oogenesis, and finally spawning and end-of-life. Up-regulation of genes involved in cellular homeostasis, glycolysis and lipid catabolism as well as germline development characterized the transition from diapause to post-diapause. The down-regulation of genes involved in cellular homeostasis coincided with the up-regulation of genes related to mid-late oogenesis and protein degradation. As females started to spawn, genes involved in protein ubiquitination and programmed cell death became up-regulated. Energy allocation and utilization is highly dynamic in N. flemingeri during the non-feeding period and it is linked to the provisioning and maturation of eggs through lipid catabolism and autophagy. The data suggest the presence of a mechanism that regulates fecundity to match the availability of stored resources. Such a mechanism would minimize reproductive failure when resources and contribute to the resilience of the species.

Exploitation of Drosophila Choriogenesis Process as a Model Cellular System for Assessment of Compound Toxicity: the Phloroglucinol Paradigm

Phloroglucinol (1,3,5 tri-hydroxy-benzene) (PGL), a natural phenolic substance, is a peroxidase inhibitor and has anti-oxidant, anti-diabetic, anti-inflammatory, anti-thrombotic, radio-protective, spasmolytic and anti-cancer activities. PGL, as a medicine, is administered to patients to control the symptoms of irritable bowel syndrome and acute renal colic, in clinical trials. PGL, as a phenolic substance, can cause cytotoxic effects. Administration of PGL up to 300 mg/kg (bw) is well tolerated by animals, while in cell lines its toxicity is developed at concentrations above the dose of 10 μg/ml. Furthermore, it seems that tumor or immortalized cells are more susceptible to the toxic power of PGL, than normal cells. However, studies of its cytotoxic potency, at the cellular level, in complex, differentiated and meta-mitotic biological systems, are still missing. In the present work, we have investigated the toxic activity of PGL in somatic epithelial cells, constituting the follicular compartment of a developing egg-chamber (or, follicle), which directs the choriogenesis (i.e. chorion assembly) process, during late oogenesis of Drosophila melanogaster. Our results reveal that treatment of in vitro growing Drosophila follicles with PGL, at a concentration of 0.2 mM (or, 25.2 μg/ml), does not lead to follicle-cell toxicity, since the protein-synthesis program and developmental pattern of choriogenesis are normally completed. Likewise, the 1 mM dose of PGL was also characterized by lack of toxicity, since the chorionic proteins were physiologically synthesized and the chorion structure appeared unaffected, except for a short developmental delay, being observed. In contrast, concentrations of 10, 20 or 40 mM of PGL unveiled a dose-dependent, increasing, toxic effect, being initiated by interruption of protein synthesis and disassembly of cell-secretory machinery, and, next, followed by fragmentation of the granular endoplasmic reticulum (ER) into vesicles, and formation of autophagic vacuoles. Follicle cells enter into an apoptotic process, with autophagosomes and large vacuoles being formed in the cytoplasm, and nucleus showing protrusions, granular nucleolus and condensed chromatin. PGL, also, proved able to induce disruption of nuclear envelope, activation of nucleus autophagy (nucleophagy) and formation of a syncytium-like pattern being produced by fusion of plasma membranes of two or more individual follicle cells. Altogether, follicle cell-dependent choriogenesis in Drosophila has been herein presented as an excellent, powerful and reliable multi-cellular, differentiated, model biological (animal) system for drug-cytotoxicity assessment, with the versatile compound PGL serving as a characteristic paradigm. In conclusion, PGL is a substance that may act beneficially for a variety of pathological conditions and can be safely used for differentiated somatic -epithelial- cells at clinically low concentrations. At relatively high doses, it could potentially induce apoptotic and autophagic cell death, thus being likely exploited as a therapeutic agent against a number of pathologies, including human malignancies.

Germ plasm-related structures in marine medaka gametogenesis; novel sites of Vasa localization and the unique mechanism of germ plasm granule arising.

Germ plasm, a cytoplasmic factor of germline cell differentiation, is suggested to be a perspective tool for in vitro meiotic differentiation. To discriminate between the: (1) germ plasm-related structures (GPRS) involved in meiosis triggering; and (2) GPRS involved in the germ plasm storage phase, we investigated gametogenesis in the marine medaka Oryzias melastigma. The GPRS of the mitosis-to-meiosis period are similar in males and females. In both sexes, five events typically occur: (1) turning of the primary Vasa-positive germ plasm granules into the Vasa-positive intermitochondrial cement (IMC); (2) aggregation of some mitochondria by IMC followed by arising of mitochondrial clusters; (3) intramitochondrial localization of IMC-originated Vasa; followed by (4) mitochondrial cluster degradation; and (5) intranuclear localization of Vasa followed by this protein entering the nuclei (gonial cells) and synaptonemal complexes (zygotene-pachytene meiotic cells). In post-zygotene/pachytene gametogenesis, the GPRS are sex specific; the Vasa-positive chromatoid bodies are found during spermatogenesis, but oogenesis is characterized by secondary arising of Vasa-positive germ plasm granules followed by secondary formation and degradation of mitochondrial clusters. A complex type of germ plasm generation, 'the follicle cell assigned germ plasm formation', was found in late oogenesis. The mechanisms discovered are recommended to be taken into account for possible reconstruction of those under in vitro conditions.

Quantitative methods for assessing local and bodywide contributions to Wolbachia titer in maternal germline cells of Drosophila

BackgroundLittle is known about how bacterial endosymbionts colonize host tissues. Because many insect endosymbionts are maternally transmitted, egg colonization is critical for endosymbiont success. Wolbachia bacteria, carried by approximately half of all insect species, provide an excellent model for characterizing endosymbiont infection dynamics. To date, technical limitations have precluded stepwise analysis of germline colonization by Wolbachia. It is not clear to what extent titer-altering effects are primarily mediated by growth rates of Wolbachia within cell lineages or migration of Wolbachia between cells.ResultsThe objective of this work is to inform mechanisms of germline colonization through use of optimized methodology. The approaches are framed in terms of nutritional impacts on Wolbachia. Yeast-rich diets in particular have been shown to suppress Wolbachia titer in the Drosophila melanogaster germline. To determine the extent of Wolbachia sensitivity to diet, we optimized 3-dimensional, multi-stage quantification of Wolbachia titer in maternal germline cells. Technical and statistical validation confirmed the identity of Wolbachia in vivo, the reproducibility of Wolbachia quantification and the statistical power to detect these effects. The data from adult feeding experiments demonstrated that germline Wolbachia titer is distinctly sensitive to yeast-rich host diets in late oogenesis. To investigate the physiological basis for these nutritional impacts, we optimized methodology for absolute Wolbachia quantification by real-time qPCR. We found that yeast-rich diets exerted no significant effect on bodywide Wolbachia titer, although ovarian titers were significantly reduced. This suggests that host diets affects Wolbachia distribution between the soma and late stage germline cells. Notably, relative qPCR methods distorted apparent wsp abundance, due to altered host DNA copy number in yeast-rich conditions. This highlights the importance of absolute quantification data for testing mechanistic hypotheses.ConclusionsWe demonstrate that absolute quantification of Wolbachia, using well-controlled cytological and qPCR-based methods, creates new opportunities to determine how bacterial abundance within the germline relates to bacterial distribution within the body. This methodology can be applied to further test germline infection dynamics in response to chemical treatments, genetic conditions, new host/endosymbiont combinations, or potentially adapted to analyze other cell and tissue types.

Transmission strategies in a chemosynthetic symbiosis: detection and quantification of symbionts in host tissues and their environment.

Transmission of bacteria vertically through host tissues ensures offspring acquire symbionts; however, horizontal transmission is an effective strategy for many associations and plays a role in some vertically transmitted symbioses. The bivalve Solemya velum and its gammaproteobacterial chemosynthetic symbionts exhibit evolutionary evidence of both transmission modes, but the dominant strategy on an ecological time scale is unknown. To address this, a specific primer set was developed and validated for the S. velum symbiont using a novel workflow called specific marker design (SMD). Symbionts were quantified in spawned eggs and sediment and seawater samples from S. velum habitats with qPCR. Each egg was estimated to contain 50-100 symbiont genomes. By contrast, symbiont DNA was found at low abundance/occurrence in sediment and seawater, often co-occurring with host mitochondrial DNA, obscuring its origin. To ascertain when eggs become infected, histological sections of S. velum tissues were labelled for symbiont 16S rRNA via in situ hybridization. This revealed symbionts in the ovary walls and mature oocytes, suggesting association in late oogenesis. These data support the hypothesis that S. velum symbionts are vertically transmitted every host generation, thus genetic signatures of horizontal transmission are driven by ecologically infrequent events. This knowledge furthers our understanding of vertical and horizontal mode integration and provides insights across animal-bacterial chemosynthetic symbioses.

Physiological characterization of the emergence from diapause: A transcriptomics approach

Organisms inhabiting high-latitude environments have evolved adaptations, such as diapause to time reproduction and growth to optimize their survival. However, the physiological regulation of the timing of complex life histories is poorly understood, particularly for marine copepods, that diapause at depth. A member of the pelagic community of the sub-Arctic Pacific Ocean, Neocalanus flemingeri enters diapause in June. Egg production occurs in winter/spring. In order to characterize the transition from diapause to egg release, females were collected in late September from 400–700 m depth, incubated in the dark at 4–5 °C and sampled for RNASeq at weekly intervals. The diapause phenotype showed down-regulation of protein turnover and up-regulation of stress genes. Activation of the reproductive program was marked by the up-regulation of genes involved in germline development. Thereafter, progress through phases of oocyte development could be linked to changes in gene expression. At 5 weeks, females showed up-regulation of spermatogenesis, indicating that stored sperm had been in a quiescent stage and completed their maturation inside the female. Gene expression profiles provide a framework to stage field-collected females. The 7-week progression from diapause to late oogenesis suggests that females typically spawning in January initiated the reproductive program in November.

Spatio-temporal patterns and reproductive costs of abnormal clutches of female American lobster, Homarus americanus, in eastern Canada

Abstract Previous studies have documented female American lobster, Homarus americanus, carrying “abnormal clutches”, i.e. with eggs covering less than half of their abdomen. From 2011 to 2014, we worked alongside harvesters to quantify spatio-temporal patterns and reproductive costs of abnormal clutches among 138 738 egg-bearing female lobsters sampled from 193 homeports spread across eastern Canada. Females with abnormal clutches were ubiquitous, being found in 90% of homeports. Their incidence was, however, relatively low, averaging 6% across sampling times and locations. The incidence decreased between spawning and 7–9 months after spawning, potentially due to cases of complete brood failure caused by sperm limitation, and it then increased toward the end of the brooding period, potentially due to repeated catch and release of ovigerous females during the fishery. In most regions, small females were more likely to carry abnormal clutches than larger females. We estimated population-level egg loss between late oogenesis and hatching of embryos at 47–51%, with approximately half being associated with normal clutches and half with abnormal clutches and complete brood failure.

The exon junction complex factor Y14 is dynamic in the nucleus of the beetle Tribolium castaneum during late oogenesis

BackgroundThe oocyte chromosomes of the red flour beetle, Tribolium castaneum, are gathered into a knot, forming a karyosphere at the diplotene stage of meiotic prophase. Chromatin rearrangement, which is a characteristic feature of oocyte maturation, is well documented. The T. castaneum karyosphere is surrounded by a complex extrachromosomal structure termed the karyosphere capsule. The capsule contains the vast majority of oocyte RNA. We have previously shown using a BrUTP assay that oocyte chromosomes in T. castaneum maintain residual transcription up to the very end of oocyte maturation. Karyosphere transcription requires evidently not only transcription factors but also mRNA processing factors, including the components of the exon junction complex with its core component, the splicing factor Y14. We employed a gene engineering approach with injection of mRNA derived from the Myc-tagged Y14 plasmid-based construct in order to monitor the newly synthesized fusion protein in the oocyte nuclei.ResultsOur preliminary data have been presented as a brief correspondence elsewhere. Here, we provide a full-length article including immunoelectron-microscopy localization data on Y14–Myc distribution in the nucleus of previtellogenic and vitellogenic oocytes. The injections of the fusion protein Y14–Myc mRNA into the oocytes showed a dynamic pattern of the protein distribution. At the previtellogenic stage, there are two main locations for the protein: SC35 domains (the analogues of interchromatin granule clusters or nuclear speckles) and the karyosphere capsule. At the vitellogenic stage, SC35 domains were devoid of labels, and Y14–Myc was found in the perichromatin region of the karyosphere, presumably at the places of residual transcription. We show that karyosphere formation is accompanied by the movement of a nuclear protein while the residual transcription occurs during genome inactivation.ConclusionsOur data indicate that the karyosphere capsule, being a destination site for a protein involved in mRNA splicing and export, is not only a specializes part of nuclear matrix separating the karyosphere from the products of chromosome activity, as believed previously, but represents a special nuclear compartment involved in the processes of gene expression in the case the karyosphere retains residual transcription activity.

MTAIL-seq reveals dynamic poly(A) tail regulation in oocyte-to-embryo development.

Eukaryotic mRNAs are subject to multiple types of tailing that critically influence mRNA stability and translatability. To investigate RNA tails at the genomic scale, we previously developed TAIL-seq, but its low sensitivity precluded its application to biological materials of minute quantity. In this study, we report a new version of TAIL-seq (mRNA TAIL-seq [mTAIL-seq]) with enhanced sequencing depth for mRNAs (by ∼1000-fold compared with the previous version). The improved method allows us to investigate the regulation of poly(A) tails in Drosophila oocytes and embryos. We found that maternal mRNAs are polyadenylated mainly during late oogenesis, prior to fertilization, and that further modulation occurs upon egg activation. Wispy, a noncanonical poly(A) polymerase, adenylates the vast majority of maternal mRNAs, with a few intriguing exceptions such as ribosomal protein transcripts. By comparing mTAIL-seq data with ribosome profiling data, we found a strong coupling between poly(A) tail length and translational efficiency during egg activation. Our data suggest that regulation of poly(A) tails in oocytes shapes the translatomic landscape of embryos, thereby directing the onset of animal development. By virtue of the high sensitivity, low cost, technical robustness, and broad accessibility, mTAIL-seq will be a potent tool to improve our understanding of mRNA tailing in diverse biological systems.