Toluidine Blue Test for Sperm DNA Integrity and Elaboration of Image Cytometry Algorithm

Background: Sperm DNA integrity is of paramount importance in the prognosis of fertility. We applied image cytometry to a toluidine blue (TB) test we recently proposed. Methods: Sperm samples from 33 men were assayed for standard sperm parameters and classified as normal or abnormal. Sperm smears were subjected to the TB test, DNA denaturation testing with acridine orange (AO), and terminal deoxyuridine triphosphate biotin nick end labeling (TUNEL). In CCD image analysis, TB-stained sperm cell heads were microscopically assigned to one of four color groups (dark, blue, light violet, and light blue). The optical densities of 6,600 cells in green and red CCD images were used to elaborate an algorithm for discrimination of these groups. Results: The proportions of sperm in TB color groups, as estimated with the developed image cytometry algorithm, correlated with microscopic features. The number of TB dark cells correlated with the number of AO-red and TUNEL+ cells. The proportion of TB dark cells in normal samples did not exceed 35%. Light-blue sperm cell heads prevailed in normal samples, whereas dark and blue sperm cell heads dominated in abnormal samples. Conclusions: The TB test was suitable for the assessment of sperm cell DNA integrity. The elaborated image cytometry algorithm can be used for this purpose and for finer determination of sperm nucleus status.publishersversionPeer reviewe

Growth control in Drosophila melanogaster

In complex multicellular organisms, growth and development has to be tightly regulated to form a fit and fertile adult. The final size of the animal is determined by two major parameters the growth rate (speed of the growth) and the duration of the juvenile growth period. The growth rate in all animals is controlled by the highly conserved insulin/insulin-like growth factor (IGF) signaling (IIS) pathway. IIS also links growth with nutrition. In Drosophila melanogaster, seven insulin-like peptides (Dilp) have been identified. Three of them are produced in specialized insulin producing cells (IPC) in the brain. The duration of the growth period is determined by the peak of a steroid hormone. However, it has been demonstrated that ecdysone, the steroid hormone controlling the developmental transitions in Drosophila, can also influence the growth rate. One of the aims of my thesis was to decipher by which physiological and molecular mechanisms ecdysone controls animal growth. We have shown that ecdysone directs the animal growth rate by specifically acting on the fat body, an organ that retains the endocrine and storage functions of the vertebrate liver and adipose tissue. We have demonstrated that loss of ecdysone receptor (EcR) function increases the expression of an oncogene dmyc as well as dMyc cellular functions such as ribosome biosynthesis. Moreover, changing the dmyc levels in this tissue is sufficient to affect the animal growth rate. Finally, we provide evidence that the dMyc level in the fat body remotely controls Dilp secretion from the IPCs. In conclusion, the present work deciphers how ecdysone signaling controls the growth rate and IIS in peripheral tissues and reveals an unexpected dMyc function in the systemic control of growth in response to steroid hormone signaling. Because IIS is tightly linked to nutrition, another aim of my thesis was to use Drosophila to elucidate how growth is controlled during the non-feeding stages of animal development. In metazoans, tissue growth relies on the availability of nutrients, either stored internally or obtained from the environment, and on the activation of IIS. In Drosophila, growth is mediated by several Dilps that act through a canonical IIS pathway. During the larval period, when animals feed, Dilps produced by the IPCs couple nutrient uptake with systemic growth. We show here that during maturation/metamorphosis, when feeding has ceased, a Dilp produced by the fat body, Dilp6, is required to relay the growth signal. Remarkably, dilp6 expression is also induced upon starvation. We further show that the expression of dilp6 during development is controlled by the steroid hormone ecdysone, and that both its developmental and environmental expression requires the forkhead box transcription factor FoxO. This study reveals a new class of insulin-like peptides induced upon metabolic stress that promote growth in conditions of nutritional deprivation or following developmentally-induced cessation of feeding. Altogether, my thesis work provides new insights into the field of growth control in Drosophila melanogaster and places the larval fat body, the functional homolog of the mammalian liver and adipose tissue, at the center of growth control.Chez les animaux multicellulaires, la formation d un adulte fertile et en bonne santé requiert un contrôle précis de la croissance. La taille finale de l animal est déterminée par deux paramètres principaux la vitesse de croissance et la durée de la période de croissance. Chez tous les animaux, la vitesse de croissance est contrôlée par la voie de signalisation de l'insuline et des facteurs de croissance apparentés à l'insuline (insulin-like growth factors - IGF). Cette voie, dite IIS (insulin/insulin-like growth factor signaling), est très conservée chez tous les animaux. De plus, la voie IIS lie croissance et nutrition. Chez la mouche Drosophila melanogaster, sept peptides apparentés à l'insuline (Drosophila insulin-like peptids, Dilps) ont été identifiés. Trois d'entre eux sont produits par des cellules du cerveau spécialisées dans la production d'insuline (insulin producing cells, IPCs). En général, la durée de la période de croissance est déterminée par un pic de concentration d'une hormone stéroïdienne. Chez la drosophile, l hormone stéroïdienne ecdysone, qui régule les transitions développementales, peut aussi influencer le taux de croissance. Un des objectifs de mon doctorat était d'élucider les mécanismes cellulaires et moléculaires par lesquels l'ecdysone contrôle la croissance. Nous avons montré que l'ecdysone contrôle la croissance en agissant directement sur le corps gras, un organe qui possède des fonctions endocrines et de stockage similaires au foie et au tissu adipeux des vertébrés. Nous avons montré qu'une perte de fonction du récepteur à l'ecdysone (EcR) augmente l'expression de l'oncogène dmyc ainsi que ses fonctions cellulaires telles que la biosynthèse des ribosomes. De plus, modifier le niveau d'expression de dmyc dans ce tissu est suffisant pour affecter le taux de croissance de l'animal. Finalement, le niveau d'expression de dmyc dans le corps gras contrôle indirectement la sécrétion de Dilp par les IPCs. En conclusion, cette étude montre comment la signalisation ecdysone contrôle le taux de croissance et l'IIS dans les tissus périphériques. De plus, nous révélons une fonction inattendue de dMyc dans le contrôle systémique de la croissance en réponse à la signalisation par les hormones stéroïdienne. La signalisation IIS étant intimement liée à la nutrition, un autre aspect de mon doctorat a consisté en l'utilisation du modèle drosophile pour étudier comment la croissance est contrôlée pendant les phases de jeûne au cours du développement. Chez les métazoaires, la croissance des tissus dépend de la disponibilité des nutriments - stockés ou obtenu à partir de l'environnement - ainsi que de l'activité de la voie IIS. Chez la drosophile, la croissance est médiée par plusieurs Dilps qui agissent via la voie IIS canonique. Pendant la période larvaire, quand les animaux se nourrissent, les Dilps produits par les IPCs associent la croissance systémique à la prise alimentaire. Nous avons montré que, pendant la maturation/métamorphose où la prise alimentaire cesse, un Dilp produit par le corps gras, Dilp6, est requis pour relayer le signal de croissance. Il est remarquable que l'expression de dilp6 soit induite par le jeûne. En outre, j'ai montré que l'hormone stéroïdienne ecdysone contrôle l'expression de Dilp6 pendant le développement et que le facteur de transcription de la famille Forkhead, FoxO, contrôle à la fois l'expression de dilp6 au cours de développement et induite par l environnement. Cette étude met à jour un nouveau peptide apparenté à l'insuline, induit par le stress métabolique et qui promeut la croissance en cas de privation alimentaire ou suite à un arrêt de la prise alimentaire lié au développement.NICE-BU Sciences (060882101) / SudocSudocFranceF

A single-cell atlas reveals unanticipated cell type complexity in Drosophila ovaries

Organ function relies on the spatial organization and functional coordination of numerous cell types. The Drosophila ovary is a widely used model system to study the cellular activities underlying organ function, including stem cell regulation, cell signaling and epithelial morphogenesis. However, the relative paucity of cell type–specific reagents hinders investigation of molecular functions at the appropriate cellular resolution. Here, we used single-cell RNA sequencing to characterize all cell types of the stem cell compartment and early follicles of the Drosophila ovary. We computed transcriptional signatures and identified specific markers for nine states of germ cell differentiation and 23 somatic cell types and subtypes. We uncovered an unanticipated diversity of escort cells, the somatic cells that directly interact with differentiating germline cysts. Three escort cell subtypes reside in discrete anatomical positions and express distinct sets of secreted and transmembrane proteins, suggesting that diverse micro-environments support the progressive differentiation of germ cells. Finally, we identified 17 follicle cell subtypes and characterized their transcriptional profiles. Altogether, we provide a comprehensive resource of gene expression, cell type–specific markers, spatial coordinates, and functional predictions for 34 ovarian cell types and subtypes.</jats:p

A transitory signaling center controls timing of primordial germ cell differentiation

Organogenesis requires exquisite spatiotemporal coordination of cell morphogenesis, migration, proliferation, and differentiation of multiple cell types. For gonads, this involves complex interactions between somatic and germline tissues. During Drosophila ovary morphogenesis, primordial germ cells (PGCs) either are sequestered in stem cell niches and are maintained in an undifferentiated germline stem cell state or transition directly toward differentiation. Here, we identify a mechanism that links hormonal triggers of somatic tissue morphogenesis with PGC differentiation. An early ecdysone pulse initiates somatic swarm cell (SwC) migration, positioning these cells close to PGCs. A second hormone peak activates Torso-like signal in SwCs, which stimulates the Torso receptor tyrosine kinase (RTK) signaling pathway in PGCs promoting their differentiation by de-repression of the differentiation gene, bag of marbles. Thus, systemic temporal cues generate a transitory signaling center that coordinates ovarian morphogenesis with stem cell self-renewal and differentiation programs, highlighting a more general role for such centers in reproductive and developmental biology

A Drosophila Insulin-like Peptide Promotes Growth during Nonfeeding States

In metazoans, tissue growth relies on the availability of nutrients—stored internally or obtained from the environment—and the resulting activation of insulin/IGF signaling (IIS). In Drosophila, growth is mediated by seven Drosophila insulin-like peptides (Dilps), acting through a canonical IIS pathway. During the larval period, animals feed and Dilps produced by the brain couple nutrient uptake with systemic growth. We show here that, during metamorphosis, when feeding stops, a specific DILP (Dilp6) is produced by the fat body and relays the growth signal. Expression of DILP6 during pupal development is controlled by the steroid hormone ecdysone. Remarkably, DILP6 expression is also induced upon starvation, and both its developmental and environmental expression require the Drosophila FoxO transcription factor. This study reveals a specific class of ILPs induced upon metabolic stress that promotes growth in conditions of nutritional deprivation or following developmentally induced cessation of feeding

Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly

For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae , that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to &gt;250 distinct cell types. We provide an in-depth analysis of cell type–related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution