Autophagy is Required for the Degeneration of the Ovarian Follicular ...

[Autophagy 2:4, 297-298; October/November/December 2006]; ©2006 Landes Bioscience

Autophagy is Required for the Degeneration of the Ovarian Follicular Epithelium in Higher Diptera Addenda

ABSTRACT Autophagy is a major pathway for the degradation of long-lived proteins and cytoplasmic organelles and an essential part of programmed cell death, as well. Our findings indicate that programmed cell death of the ovarian follicle cells in the higher Diptera species Bactrocera oleae and Ceratitis capitata manifests features of autophagic cell death. The follicle cells during the developmental stage 14 contain autophagic vacuoles and they do not exhibit caspase activity in any area of the egg chamber. Their nuclei are characterized by condensed chromatin, accompanied with high-but not low-molecular weight DNA fragmentation events exclusively detected in distinct cells of the anterior pole. The above results are likely associated with the abundant phagocytosis observed at the entry of the lateral oviducts, where numerous cell bodies are massively engulfed by epithelial cells. The similarity of the cell death process among B. oleae, C. capitata and Drosophila melanogaster species strongly suggests that autophagy-mediated cell death is conserved in higher Diptera species.

Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/abstract.php?id=2858

KEY WORDS autophagosomes, autophagy, Diptera, Drosophila, follicle cells, phagocytosis, programmed cell death

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The structural and functional unit of an insect’s ovary is the egg chamber, or else, the follicle. In the meroistic polytrophic ovarioles of higher Diptera, including Drosophila melanogaster, each egg chamber consists of 16 germ line cells (15 nurse cells and 1 oocyte), surrounded by a monolayer of somatic epithelial cells called follicle cells.1-2 The follicle cells differentiate into distinct subpopulations, which move to specific positions of the egg chamber, participate in oocyte polarity formation and during the late stages of oogenesis secrete the eggshell, or chorion.1,3-4 When the egg chamber completes the process of oogenesis, at the final developmental stage 14, it exits the ovariole and passes through the lateral and the common oviduct. Contraction of the oviduct forces the egg into the uterus, where fertilization occurs.5 Programmed cell death constitutes a highly conserved, genetically regulated process, implicated in a wide variety of different biological systems, including insect’s oogenesis, that leads to the self-destruction of superfluous cells, through the activation of a cell suicide program.6 Programmed cell death includes two major types, apoptotic cell death and autophagic cell death. Apoptotic cell death is mainly characterized by chromatin condensation, DNA fragmentation and activation of caspases, whereas there is no evidence of major ultrastructural changes in the cytoplasmic organelles. In the autophagy-mediated cell death, the accumulation of autophagic vacuoles in the cytoplasm represents the most distinct morphological feature of this type of programmed cell death,7-9 whereas the execution of the autophagic cell death program does not seem to require the participation of caspases.10-11 In both cases, the dying cells are phagocytosed by phagocytes.12 In a recent study, we have tried to elucidate the distinct type of programmed cell death of the follicular epithelium during late oogenesis in the higher Diptera species Bactrocera oleae and Ceratitis capitata.13 Our findings demonstrate that at the end of oogenesis, in both Diptera species, ovarian follicle cells, which are post-mitotic cells, exhibit prominent features of autophagic cell death. The follicle cells of stage 14 egg chambers contain characteristic autophagic vacuoles filled with cytoplasmic material and various organelles, such as mitochondria, ribosomes, pieces of cytoplasm and rough endoplasmic reticulum membranes and tightly packed, concentric membrane whorls (Fig. 1A and B).13 The presence of autophagic vacuoles and autolysosomes is also detected in the follicular epithelium of D. melanogaster, as well (Fig. 1C and D). By using monodansylcadaverine staining, several positive signals are detected in the follicular epithelium of the main body, anterior and posterior pole of the developing egg chambers, in both species.

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ACKNOWLEDGEMENTS

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Received 5/2/06; Accepted 5/3/06

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*Correspondence to: Issidora S. Papassideri; Faculty of Biology; Department of Cell Biology and Biophysics; University of Athens; Panepistimiopolis, Athens 15784 Greece; Tel.: +30.210.727.4546; Fax: +30.210.727.4742; Email: ipapasid@ biol.uoa.gr

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Faculty of Biology; Department of Cell Biology and Biophysics; University of Athens; Athens, Greece

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Ioannis P. Nezis Dimitrios J. Stravopodis Lukas H. Margaritis Issidora S. Papassideri*

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I.P. Nezis is a scholarship recipient of the Hellenic State Scholarship Foundation (IKY) (Post-doctoral fellowship).

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Addendum to:

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Programmed Cell Death of Follicular Epithelium During the Late Developmental Stages of Oogenesis in the Fruit Flies Bactrocera oleae and Ceratitis capitata (Diptera, Tephritidae) is Mediated by Autophagy

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I.P. Nezis, D.J. Stravopodis, L.H. Margaritis and I.S. Papassideri Dev Growth Differ 2006; 48:189-98

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Autophagy-Mediated Cell Death in Ovarian Follicle Cells

Figure 1. The ovarian follicular epithelium at stage 14 of oogenesis in Bactrocera oleae, Ceratitis capitata and Drosophila melanogaster Diptera species contains characteristic autophagic vacuoles and autolysosomes. (A) Transmission electron micrograph of a stage 14 C. capitata egg chamber, demonstrating a representative follicle cell at the main body containing an autophagic vacuole (arrow). It has enveloped various cytoplasmic components, such as small vacuoles, portions of endoplasmic reticulum, ribosomes, multi-lamellar membranes and a mitochondrion (small arrow). (B) Transmission electron micrograph of a stage 14 B. oleae egg chamber, revealing a representative follicle cell at the posterior pole with a predominant occurrence of an autolysosome (arrow). (C) Transmission electron micrograph of a stage 14 D. melanogaster egg chamber at the main body, exhibiting the presence of several autophagic vacuoles/ autolysosomes (arrows). (D) Transmission electron micrograph of a stage 14 D. melanogaster egg chamber at the anterior pole, indicating a characteristic autolysosome. It mainly contains pieces of cytoplasm (arrow) and tightly packed, concentric multi-lamellar membranes (arrowheads). FC, follicle cell; Ch, chorion. Scale bars: 1 µm.

The follicle cells do not exhibit any detectable signal of caspase activity, as revealed by an active caspase in situ assay. The nuclei of the follicle cell subpopulations located at the anterior pole are the only ones that are characterized by condensed chromatin, without containing low molecular weight DNA fragmentation, as disclosed by the absence of TUNEL labeling. On the contrary, acridine orange staining demonstrates the occurrence of high molecular weight DNA fragmentation events in several follicle cell nuclei of the anterior pole. The fact that the alterations in follicle cell nuclear morphology are exclusively observed at the anterior pole of the egg chamber strongly suggests that these subpopulations enter early in the final stage of their autophagic cell death, in contrast to the ones located on other regions of the follicle, which likely remain at an initial execution phase of their cell death program. This position-dependant follicle cell autophagic death could be associated with the spatial pattern of chorion synthesis, which is first completed at the anterior pole of the egg chamber, during late oogenesis, in all Diptera species studied so far.1,14 Thus, in both B. oleae and C. capitata flies, the ovarian follicle cells at the anterior pole, after having fulfilled their biological role in egg chamber maturation, follow a temporal- and spatial-specific cell death program, resulting in the degeneration of the follicular epithelium. Similar features have been also observed during late oogenesis in D. melanogaster (Diptera, Drosophilidae)15 and Acdyonurus venosus (Ephemeroptera, Heptageniidae),16 where 298

distinct follicle cell subpopulations of the one pole exhibit characteristic signs of programmed cell death. After the completion of chorion synthesis, the egg chambers pass through and exit from the ovariole to the lateral oviducts, when contractions of the epithelial sheath cells occur. These contractions likely force the degenerated follicle cell layer to detach from the chorion of both B. oleae and C. capitata species. We asked where the degenerated follicle cells are localized after their detachment from chorion. Our study reveals that abundant phagocytosis is restricted at the entry of the lateral oviducts, as revealed by acridine orange staining and examination of the respective areas of the oviduct by light and transmission electron microscopy. The phagocytosed cellular debris, observed within the epithelial cells of the oviducts, are likely derived from the degenerated follicle cell layer undergoing autophagic cell death. Thus, we assume that the degenerated follicle cells are efficiently phagocytosed by the epithelial cells at the entry of the lateral oviducts, preventing the blockage of the ovarioles, which occurs if the accumulated cellular remnants are not properly absorbed. In conclusion, programmed cell death of the follicular epithelium appears to be remarkably similar among B. oleae, C. capitata and D. melanogaster species,13,15 manifesting prominent features of autophagic cell death. Thus, we suggest that the developmentally regulated degeneration of the ovarian follicular epithelium is a phylogenetically conserved biological process among higher Diptera species. References 1. Trougakos IP, Margaritis LH. Novel morphological and physiological aspects of insect eggs. In: Hilker M, Meiners T, eds. Chemoecology of Insect Eggs and Egg Deposition. Berlin, Germany: Blackwell Wissenschaftsverlag, 2002:3-36. 2. Berg CA. The Drosophila shell game: Patterning and morphological change. Trends Genet 2005; 21:346-55. 3. Margaritis LH. Structure and physiology of the eggshell. In: Gilbert LI, Kerkut GA, eds. Comprehensive Insect Biochemistry, Physiology and Pharmacology Vol 1. Oxford, New York: Pergammon Press, 1985:151-230. 4. Margaritis LH, Mazzini M. Structure of the egg. In: Harrison FW, Locke M, eds. Microscopic Anatomy of Invertebrates, Vol 11C, “Insecta”. New York: Wiley-Liss Publ, 1998:995-1037. 5. Bloch Qazi MC, Heifetz Y, Wolfner MF. The development between gametogenesis and fertilization: Ovulation and female sperm storage in Drosophila melanogaster. Dev Biol 2003; 256:195-211. 6. Danial NN, Korsmeyer SJ. Cell death: Critical control points. Cell 2004; 116:205-19. 7. Bursch W. Multiple cell death programs: Charon’s lifts to Hades. FEMS Yeast Res 2004; 5:101-10. 8. Lockshin RA, Zakeri Z. Apoptosis, autophagy and more. Int J Biochem Cell Biol 2004; 36:2405-19. 9. Yorimitsu T, Klionsky DJ. Autophagy: Molecular machinery for self-eating. Cell Death Differ 2005; 12:1542-52. 10. Bursch W. The autophagosomal-lysosomal compartment in programmed cell death. Cell Death Differ 2001; 8:569-81. 11. Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene 2004; 23:2891-906. 12. Krieser RJ, White K. Engulfment mechanism of apoptotic cells. Curr Opin Cell Biol 2002; 14:734-8. 13. Nezis IP, Stravopodis DJ, Margaritis LH, Papassideri IS. Programmed cell death of follicular epithelium during the late developmental stages of oogenesis in the fruit flies Bactrocera oleae and Ceratitis capitata (Diptera, Tephritidae) is mediated by autophagy. Dev Growth Differ 2006; 48:189-98. 14. Margaritis LH, Kafatos FC, Petri WH. The eggshell of Drosophila melanogaster I: The fine structure of the layers and regions of the wild type eggshell. J Cell Sci 1980; 43:1-35. 15. Nezis IP, Stravopodis DJ, Papassideri IS, Robert-Nicoud M, Margaritis LH. The dynamics of apoptosis in the ovarian follicle cells during the late stages of Drosophila oogenesis. Cell Tissue Res 2002; 307:401-9. 16. Gaino E, Rebora M. Detection of apoptosis in the ovarian follicle cells of Acdyonurus venosus (Ephemeroptera, Heptageniidae). Ital J Zool 2003; 70:291-5.

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2006; Vol. 2 Issue 4