Dataset Information

Three Dimensional Secondary Ion Mass Spectrometry Imaging (3D-SIMS) of Aedes aegypti ovarian follicles.

ABSTRACT: The mobilization of nutrient reserves into the ovaries of Aedes aegypti mosquitoes after sugar-feeding plays a vital role in the female's reproductive maturation. In the present work, three-dimensional secondary ion mass spectrometry imaging (3D-SIMS) was used to generate ultrahigh spatial resolution (~1 ?m) chemical maps and study the composition and spatial distribution of lipids at the single ovarian follicle level (~100 ?m in size). 3D-Mass Spectrometry Imaging (3D-MSI) allowed the identification of cellular types in the follicle (oocyte, nurse and follicular cells) using endogenous markers, and revealed that most of the triacyglycerides (TGs) were compartmentalized in the oocyte region. By comparing follicles from water-fed and sugar-fed females (n=2), 3D-MSI-Time of Flight-SIMS showed that TGs were more abundant in ovarian follicles of sugar-fed females; despite relative sample reproducibility per feeding condition, more biological replicates will better support the trends observed. While the current 3D-MSI-TOF-SIMS does not permit MS/MS analysis of the lipid species, complementary LC-MS/MS analysis of the ovarian follicles aided tentative lipid assignments of the SIMS data. The combination of these MS approaches is giving us a first glimpse of the distribution of functionally relevant ovarian lipid molecules at the cellular level. These new tools can be used to investigate the roles of different lipids on follicle fitness and overall mosquito reproductive output.

SUBMITTER: Castellanos A

PROVIDER: S-EPMC6824543 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Three Dimensional Secondary Ion Mass Spectrometry Imaging (3D-SIMS) of <i>Aedes aegypti</i> ovarian follicles.

Castellanos Anthony A Ramirez Cesar E CE Michalkova Veronika V Nouzova Marcela M Noriega Fernando G FG Francisco Fernández-Lima FL

Journal of analytical atomic spectrometry 20190221 5

The mobilization of nutrient reserves into the ovaries of <i>Aedes aegypti</i> mosquitoes after sugar-feeding plays a vital role in the female's reproductive maturation. In the present work, three-dimensional secondary ion mass spectrometry imaging (3D-SIMS) was used to generate ultrahigh spatial resolution (~1 μm) chemical maps and study the composition and spatial distribution of lipids at the single ovarian follicle level (~100 μm in size). 3D-Mass Spectrometry Imaging (3D-MSI) allowed the id ...[more]

PMID: 31680712

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Project description:Various toxicants, drugs and their metabolites carry potential ovarian toxicity. Ovarian follicles, the functional unit of the ovary, are susceptible to this type of damage at all stages of their development. However, despite of the large scale of potential negative impacts, assays that study ovarian toxicity are limited. Exposure of cultured ovarian follicles to toxicants of interest served as an important tool for evaluation of toxic effects for decades. Mouse follicles cultured on the bottom of a culture dish continue to serve an important approach for mechanistic studies. In this paper, we demonstrated the usefulness of a hydrogel based 3-dimensional (3D) mouse ovarian follicle culture as a tool to study ovarian toxicity in a different setup. The 3D in vitro culture, based on fibrin alginate interpenetrating network (FA-IPN), preserves the architecture of the ovarian follicle and physiological structure-function relationship. We applied the novel 3D high-throughput (HTP) in vitro ovarian follicle culture system to study the ovotoxic effects of an anti-cancer drug, Doxorobucin (DXR). The fibrin component in the system is degraded by plasmin and appears as a clear circle around the encapsulated follicle. The degradation area of the follicle is strongly correlated with follicle survival and growth. To analyze fibrin degradation in a high throughput manner, we created a custom MATLAB® code that converts brightfield micrographs of follicles encapsulated in FA-IPN to binary images, followed by image analysis. We did not observe any significant difference between manually processed images to the automated MATLAB® method, thereby confirming that the automated program is suitable to measure fibrin degradation to evaluate follicle health. The cultured follicles were treated with DXR at concentrations ranging from 0.005 nM to 200 nM, corresponding to the therapeutic plasma levels of DXR in patients. Follicles treated with DXR demonstrated decreased survival rate in greater DXR concentrations. We observed partial follicle survival of 35% ± 3% (n = 80) in 0.01nM treatment and 48% ± 2% (n = 92) in 0.005nM, which we identified as the IC50 for secondary follicles. In summary, we established a 3D in vitro ovarian follicle culture system that could be used in an HTP approach to measure toxic effects on ovarian follicles.

Project description:BackgroundInsects live in almost every habitat on earth. To adapt to their diverse environments, insects have developed a myriad of different strategies for reproduction reflected in diverse anatomical and behavioral features that the reproductive systems of females exhibit. Yet, ovarian development remains largely uncharacterized in most species except Drosophila melanogaster (D. melanogaster), a high Diptera model. In this study, we investigated the detailed developmental process of the ovary in Aedes aegypti (Ae. aegypti), a major vector of various disease-causing pathogens that inhabits tropical and subtropical regions.ResultsCompared with Drosophila melanogaster, a model of higher Diptera, the processes of pole cell formation and gonad establishment during embryonic stage are highly conserved in Ae. aegypti. However, Ae. aegypti utilizes a distinct strategy to form functional ovaries during larval/pupal development. First, during larval stage, Ae. aegypti primordial germ cells (PGCs) undergo a cyst-like proliferation with synchronized divisions and incomplete cytokinesis, leading to the formation of one tightly packed "PGC mass" containing several interconnected cysts, different from D. melanogaster PGCs that divide individually. This cyst-like proliferation is regulated by the target of rapamycin (TOR) pathway upon nutritional status. Second, ecdysone-triggered ovariole formation during metamorphosis exhibits distinct events, including "PGC mass" breakdown, terminal filament cell degeneration, and pre-ovariole migration. These unique developmental features might explain the structural and behavioral differences between Aedes and Drosophila ovaries. Importantly, both cyst-like proliferation and distinct ovariole formation are also observed in Culex quinquefasciatus and Anopheles sinensis, suggesting a conserved mode of ovarian development among mosquito species. In comparison with Drosophila, the ovarian development in Aedes and other mosquitoes might represent a primitive mode in the lower Diptera.ConclusionsOur study reveals a new mode of ovarian development in mosquitoes, providing insights into a better understanding of the reproductive system and evolutionary relationship among insects.

Dataset Information

Three Dimensional Secondary Ion Mass Spectrometry Imaging (3D-SIMS) of Aedes aegypti ovarian follicles.

Publications

Three Dimensional Secondary Ion Mass Spectrometry Imaging (3D-SIMS) of <i>Aedes aegypti</i> ovarian follicles.

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