Ralph G. Thomé, Helio B. Santos, Fabio P. Arantes, Fabrício F.T. Domingos, Nilo Bazzoli and Elizete Rizzo*
Departamento de Morfologia; Instituto de Ciências Biológicas; Universidade Federal de Minas Gerais; Belo Horizonte, Brasil
Key words: autophagy, apoptosis, follicular atresia, follicular cell, ovary, teleost fish
[Autophagy 5:1, 117-119; 1 January 2009]; ©2009 Landes Bioscience
*Correspondence to: Elizete Rizzo; Departamento de Morfologia; Instituto de Ciências Biológicas; Universidade Federal de Minas Gerais; UFMG; C. P.486; Belo Horizonte 30161-970 MG, Brasil; Tel.: +553134092785; Fax: +553134092771; Email: ictio@icb.ufmg.br
Submitted: 10/03/08; Revised: 10/22/08; Accepted: 10/30/08 Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/article/7302
Addendum to: Santos HB, Thomé RG, Arantes FP, Sato Y, Bazzoli N, Rizzo E. Ovarian follicular atresia is mediated by heterophagy, autophagy and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes). Theriogenology 2008; 70:1449–60; PMID: 18701155; DOI: 10.1016/j.theriogenology.2008.06.091.
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Autophagy during follicular atesia in fish ovary118 Autophagy 2009; Vol. 5 Issue 1
and rainbow trout indicated that apoptosis could not be a prominent early event in the onset of follicular atresia.18,19
In our studies, apoptosis has been detected in the follicular cells by the TUNEL assay that showed DNA fragmentation (Fig. 1B), and also by other major morphological hallmarks of apoptosis (Fig 1C): chromatin condensation in a crescent pattern underlying the nuclear envelope, cell shrinkage, loss of cell-cell interactions, detachment from basement membrane, and cell fragmentation in apoptotic bodies. The ladder pattern of the apoptotic DNA was also revealed by agarose gel electrophoresis (Fig 1G) during postspawning regression in P. argen-
teus ovaries.16 Through morphometry, we recently
detected an apoptosis rate (%) increasing progres- sively in follicular cells of postovulatory follicles until three days and decreasing thereafter.14,16 In contrast,
follicular apoptosis increased only during late follic- ular atresia, which occurred about four to six months after breeding.17 Thus, we believe that apoptosis
contributes greatly to the removal of follicular cells in postovulatory follicles, but alternative mechanisms of PCD could also be active in the follicular atresia.
Autophagic vacuoles with degenerating organelles were abundant in follicular cells (Fig. 1D and E) during yolk removal in P. argenteus and L. taeniatus.17 Large vacuoles filled with cytoplasmic material
and organelles, such as mitochondria, ribosomes and endoplasmic reticulum are features of macroautophagy, which is associated with autophagic or type II PCD.3,6 Multivesicular bodies and
myelin-like structures (Fig. 1F) are also commonly detected during follicular atresia in teleosts by electron microscopy.13,17 In support
of a prodeath function for autophagy during follicular atresia, we have detected immunohistochemical labeling for caspase 3 in the follicular cells of postovulatory follicles of P. argenteus, whereas atretic follicles were not labeled during advanced regression.17 The absence
of caspase 3 activation is usually viewed as a requisite in order to categorize an alternative cell death pathway.4 In mammals and birds,
studies on follicular atresia have focused on the apoptosis of granu- losa cells, but autophagic cell death has not been ruled out.20 Both types of PCD have also been reported to act cooperatively to effi- ciently eliminate the degenerated nurse cell during Drosophila virilis
Figure 1. Ovarian regression in Prochilodus argenteus and
Leporinus taeniatus. (A) Yolk (Y) and zona pellucida (ZP)
degeneration and engulfment (arrow) by hypertrophied fol- licular cells (FC) during follicular atresia. (B) TUNEL-positive reaction in follicular cells and apoptotic body (arrowhead). (C) Follicular cell undergoing apoptosis with loss of cell adhesion (asterisk), surface blebbing (B) and nuclear fragmentation (double arrow) in postovulatory follicle. (D) Follicular cell undergoing intense autophagic activity with lysosomes (L) and autophagic vacuoles (AV) with degen- erating organelles. (E) Large autophagic vacuole with cytoplasmic material and degenerating mitochondria (M). (F) Multivesicular body (MB) and myelin-like structure (MF) in follicular cell. (G) Ladder pattern of apoptotic DNA by agarose gel eletrophoresis in different times after spawn- ing. Scale bars: (A and B): 10 μm, (C and D): 1 μm, (E and F): 0.5 μm.
oogenesis.21 In addition to a probable contribution in PCD,
autophagy is primarily a survival mechanism in starving cells usually utilized to generate both nutrients and energy for maintaining cell viability.4 Regarding follicular atresia in fish ovary, we believe that
autophagy might have a prosurvival role for follicular cells as long as they are actively involved in oocyte removal. The interaction of Beclin 1 with the antiapoptotic protein Bcl-2 is crucial in determining cell fate and the crosstalk between autophagy and apoptosis.22 However,
the relationship among the PCD types still needs to be investigated in fish ovary after spawning.
In conclusion, we propose a hypothesis for follicular cell removal during ovarian regression in oviparous fish (Fig. 2). In this model, autophagy could play dual roles in response to follicular atresia.
Acknowledgements
We are grateful to Dr. Yoshimi Sato for his valuable contribu- tion during experimental procedures in Hydrobiology and Hatchery Station of Três Marias-CEMIG GT/CODEVASF. The work was supported by grants from FAPEMIG, CNPq and CAPES.
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Autophagy during follicular atesia in fish ovarywww.landesbioscience.com Autophagy 119
21. Velentzas AD, Nezis IP, Stravopodis DJ, Papassideri IS, Margaritis LH. Mechanisms of programmed cell death during oogenesis in Drosophila virilis. Cell Tissue Res 2007; 327:399-414.
22. Klionsky DJ. Autophagy: from phenomenology to molecular understanding in less than a decade. Nat Rev Mol Cell Biol 2007; 8:931-7.
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Figure 2. Hypothesis for the follicular cell fate in ovipa- rous fish ovary after breeding. In the postovulatory folli- cle, apoptosis or type I cell death is the major cell death pathway, which contributes greatly to follicular cell removal, although possible involvement of autophagy is not rejected. In the follicular atresia, autophagy is the primary response in order to maintain viability of the follicular cell. However, some cells may also com- mit to die by apoptosis, independent or dependent- caspase. Another role in cell clearance is also possible for autophagy, when phagocytes are not sufficient for removal of the whole cell remains. The crosstalk between apoptosis and autophagy would have a cytoprotective effect leading to survival of the follicular cell during oocyte deletion. Autophagic vacuoles accu- mulated in follicular cells during late atresia induce autophagy-mediated or type II cell death. Otherwise, apoptosis could be triggered for more efficient removal of the follicular cells in follicular atresia.
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