Apoptosis and autophagy-related gene transcription during ovarian follicular atresia in European hake (Merluccius merluccius) (original) (raw)
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Cell and Tissue Research, 2012
Follicular atresia in fish ovary provides an interesting model for studying autophagy and apoptosis. In order to improve knowledge of the mechanisms regulating ovarian regression, we investigated the immunolocalisation of various proteins involved in the complex network of autophagy and apoptosis. Females of three species of freshwater fish maintained in captivity were sampled after the reproductive period and the main events of follicular atresia were assessed by histology: splits in the zona radiata, yolk degradation and reabsorption, hypertrophy of the follicular cells, accumulation of autophagic vacuoles, closing of the follicular lumen and thickening of the theca. The interplay of apoptosis and autophagy was analysed by TUNEL in situ and by immunocytochemistry for caspase-3, bax, bcl-2, beclin-1 and cathepsin-D. During early and advanced stages of follicular regression, the actin cytoskeleton was well developed and labelling for bcl-2 and cathepsin-D were pronounced in the follicular cells at a stage when they were intensively involved in yolk phagocytosis. Immunofluorescence for beclin-1 was prevalent in the follicular cells, punctate labelling often surrounding autophagic vacuoles during the advanced stage of follicular regression, a critical step towards cell death. TUNEL-positive reaction and immunostaining for bax and caspase-3 demonstrated the participation of apoptosis in late follicular regression. Overall, this study provides evidence that autophagic and apoptotic proteins are activated in a coordinated fashion depending on the stage of follicular regression, with interplay between autophagy and apoptosis being essential in determining the fate of the cell during follicular atresia in fish ovary.
Dual roles for autophagy during follicular atresia in fish ovary
Autophagy
Autophagy, a highly conserved catabolic program for degrading proteins and organelles, is essential for cell and tissue homeostasis. Primarily, this process has a cytoprotective role under nutrient deprivation, but several stress stimuli can induce autophagy and, thus, distinct programmed cell death (PCD) pathways can be actived when stress is not abolished. Fish ovaries are a suitable experimental model system for studying the mechanisms of PCD due to the presence of postovulatory and atretic (i.e., nonovulated) follicles, which follow different routes after spawning. Apoptosis of the follicular cells is the major mechanism responsible for the rapid resorption of the postovulatory follicles. Recently, we investigated the contribution of PCD during follicular atresia in two species of freshwater fish. In contrast to mammals, this study revealed that follicular apoptosis is not a major process for deletion of follicular cells in atretic follicles. Furthermore, we detected autophagic ...
Molecular Reproduction and Development, 2019
Follicular atresia is a hormonally controlled degenerative process involving apoptosis of the somatic and germ cells. Since different signaling pathways can induce cell death, the aim of the present study was to investigate cell death signaling and crosstalk between autophagic, apoptotic, and lysosomal proteins during follicular atresia in Nile tilapia. For this, females were kept in controlled conditions for 21 days, and ovary samples were collected weekly. The atretic follicles (AF) were analyzed in three regression phases: Early, advanced, and late. Under electron microscopy, the follicular cells exhibited numerous protein synthesis organelles in the early AF. Immunoreactivity for Bcl2, Beclin1, Lc3, and Cathepsin D increased significantly in advanced AF (p < .001), when follicular cells were in intense yolk phagocytosis. In this phase, autophagosomes and autolysosomes were frequently observed. In the late AF, follicular cells had a markedly electron-lucid cytoplasm and immunoreactivity for Bax and TUNEL assay indicated an elevated apoptosis rate. Colocalisation of Lamp1/ Cathepsin D and Lc3/Caspase-3 suggests dynamic crosstalk between the autophagy, apoptosis, and lysosome pathways. Taken together, the data indicate that autophagy plays a role in the homeostasis and clearance of the follicular cells preceding Cathepsin D mediated apoptosis during follicular atresia in Nile tilapia.
Theriogenology, 2008
We investigated apoptosis, cell proliferation antigen (PCNA), and heat shock protein (HSP70) during ovarian follicular atresia in two freshwater teleost species from the São Francisco River basin, Brazil: curimatã-pacu, Prochilodus argenteus and piau-jejo, Leporinus taeniatus. Fishes were maintained in captivity after the reproductive period and ovarian regression was assessed by gonadosomatic index for three stages: early, advanced, and late regression. Follicular atresia was analysed by light and transmission electron microscopy, as well as by TUNEL and immunohistochemistry for HSP70 and PCNA. During early regression, atretic follicles exhibited zona pellucida breakdown, yolk degeneration, and hypertrophied follicular cells (e.g. granulosa in mammals). Intense heterophagy to engulf the yolk, and autophagy were detected in the follicular cells during advanced and late atresia. The TUNEL assay detected DNA fragmentation, mainly in late follicular atresia. The apoptosis rate of the follicular cells increased up to 10% during follicular atresia in both species and was negatively correlated with follicular area. Immunohistochemistry reaction for HSP70 stained the follicular cells strongly during advanced atresia, when they are intensively involved in yolk engulfment, whereas the reaction for PCNA labelled theca cells. We inferred that heterophagy, autophagy, and apoptosis contributed to follicular atresia in teleost ovaries, thereby achieving a more efficient removal of the degenerating oocyte and dying follicular cells. Additionally, HSP70 may protect the follicular cells before apoptosis when they are involved in yolk engulfment, and cell proliferation in the theca contributed to ovarian remodelling. #
Tissue and Cell, 2012
Aiming to better understand folliculogenesis, this study evaluated cell death and proliferation of ovarian cells, besides cathepsin-D expression in Prochilodus argenteus captured in two sites of the São Francisco River downstream from the Três Marias Dam, Brazil. In the site immediately following the Dam (S1), low levels of dissolved oxygen were registered in the rainy period. The water temperature was higher in the São Francisco River immediately after the confluence with the Abaeté River (S2), regardless of the period. In S1, the ovaries showed smaller oocytes, high caspase-3 enzymatic activity and apoptosis, lower cells in proliferation and GSI, as well as a lesser quantity of cathepsin-D when compared to females captured from S2. Regarding relative frequency of ovarian structures, in the dry period, only oogonia and perinucleolar oocytes were found in fish ovaries from both sites. On the other hand, in the rainy period, the relative frequency of oogonia and perinucleolar oocytes decreased and the vitellogenic oocytes increased in S2. Postovulatory follicles were observed only in S2, whereas atretic follicles occurred at a higher frequency in S1. Our results showed that apoptosis, cell proliferation and cathepsina-D evaluation can be used as biomarkers of environmental impact.
BMC Genomics, 2009
The Senegalese sole (Solea senegalensis) is a marine flatfish of increasing commercial interest. However, the reproduction of this species in captivity is not yet controlled mainly because of the poor knowledge on its reproductive physiology, as it occurs for other non-salmonid marine teleosts that exhibit group-synchronous ovarian follicle development. In order to investigate intra-ovarian molecular mechanisms in Senegalese sole, the aim of the present study was to identify differentially expressed genes in the ovary during oocyte growth (vitellogenesis), maturation and ovarian follicle atresia using a recently developed oligonucleotide microarray.
Atresia and apoptosis in preovulatory follicles in the ovary of Danio rerio (Zebrafish)
Turkish Journal of Fisheries and Aquatic Sciences, 2009
Although apoptosis, the programmed cell death, is the main mechanism in follicular atresia and especially postovulatory regression in mammals, its role in fish ovary is still controversial. In order to discuss the possible functions of atresia as a context of apoptosis, the preovulatory follicles of the freshwater teleost, Danio rerio (zebrafish) were investigated by light microscopy. The main characteristics of atresia of vitellogenic oocytes of zebrafish were determined as a significant hypertrophy and hyperplasia of the granulosa cells. The theca cells also became slightly hyperplasic while the zona radiata was degenerated and the basal membrane located between granulosa and theca was also disintegrated. The aggregation of compacted chromatin in a crescent pattern, and cell fragmentation that indicates to formation of apoptotic bodies were evaluated as the morphological evidences for apoptosis in granulosa cells of the oocytes of zebrafish. Towards the end of atresia, the vitellus is fully degenerated and absorbed, some apoptotic bodies were also observed between the translucent clusters of theca cells. Apoptotic process was still continued when an atrium had been formed.
Reproduction (Cambridge, England), 2006
It has been widely accepted that mammalian females are born with a non-renewing, finite pool of oocytes that will be continuously cleared by atresia, with only a small proportion of them reaching ovulation. Apoptosis regulates this mass germ cell death, especially through the balance between pro-and anti-apoptotic proteins encoded by the BCL-2 gene family. The caviomorph rodent Lagostomus maximus, the South American plains viscacha, displays the highest ovulation rate known for a mammal releasing 400-800 eggs per cycle. We tested the hypothesis that in L. maximus massive polyovulation is a consequence of reduced apoptosis resulting in suppressed follicular atresia. We found that anti-apoptotic BCL-2 gene is markedly expressed in all kind of follicles from primordial to fully mature antral stages in the adult ovary of L. maximus. On the other hand, pro-apoptotic BAX gene showed weak signals or was undetectable by immunohistochemical examination. Western blot against both proteins confirmed immunohistochemical results. Screening for DNA fragmentation by TUNEL assay was conspicuously negative in ovaries from both pregnant and non-pregnant females. In addition, a-oestrogen receptor also showed an enhanced expression from primordial stage to fully mature antral follicles. Our results show that natural preferential expression of BCL-2 and restricted BAX expression greatly suppresses apoptosis in the ovary of L. maximus. This prevents the decline of the oocyte reserve by abolishing follicular atresia and enables the highest ovulation rate known for a mammal, 400-800 or more eggs per cycle.
General and Comparative Endocrinology, 1997
In this study we present the first evidence for the occurrence of apoptotic cell death in ovarian follicles from teleost fish. Preovulatory ovarian follicles from mature hatchery-raised rainbow trout (Oncorhynchus mykiss) were collected and either immediately frozen in liquid nitrogen or incubated in serum-free medium at 187 for 24 hr. The extent of ovarian apoptotic DNA fragmentation was determined using 38-end labeling of DNA with [ 32 P]dideoxy-ATP, size fractionation by agarose gel electrophoresis, and quantification of low-molecular-weight (F15 kb) DNA using autoradiography and liquid scintillation counting. The extent of apoptotic DNA fragmentation was eightfold greater in immediately frozen preovulatory follicles than in previtellogenic ovarian follicles collected from immature rainbow trout (P F 0.05), suggesting differences in the degree of apoptosis at different stages of follicular development. In preovulatory trout follicles, the extent of apoptotic DNA fragmentation was fivefold greater in follicles incubated for 24 hr. Treatment of incubated preovulatory follicles with either partially purified salmon gonadotropin SG-G100 (1 mg/ml) or epidermal growth factor (EGF; 100 ng/ml) suppressed apoptotic DNA fragmentation by 31 and 41%, respectively, in comparison to untreated incubated follicles (P F 0.01). Treatment of incubated follicles with 17bestradiol (1-100 ng/ml) caused a concentration-dependent suppression of apoptotic DNA fragmentation (P F 0.05). These results suggest that apoptosis is involved in teleost ovarian development and that several of the hormonal factors acting as follicle survival factors in mammalian and avian ovaries may play a similar role in teleost ovarian follicles. r 1997 Academic Press
Apoptosis and Ovarian Function: Novel Perspectives from the Teleosts
Biology of Reproduction, 2001
Apoptosis is a fundamental mechanism in follicular atresia and postovulatory regression in mammals, but its role in teleost ovarian function is currently unknown. This study tested the hypotheses that apoptosis mediates follicular atresia in teleosts and is inducible in vitro by incubation in serum-free conditions. Vitellogenic follicles from rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus) were incubated overnight in serum-free medium and examined for apoptosis by 3-end-labeling and/or TUNEL analysis. Primary, postovulatory, and oocytectomized vitellogenic trout follicles and atretic goldfish follicles were evaluated in similar fashion. Overall, goldfish follicles had lower levels of DNA fragmentation than trout follicles. The DNA fragmentation in atretic goldfish follicles was similar to that measured in healthy vitellogenic and prematurational follicles; DNA fragmentation did not change after incubation. In the trout, postovulatory and oocytectomized vitellogenic follicles showed significantly greater in vitro susceptibility to apoptosis than intact vitellogenic follicles, whereas primary follicles were least susceptible. The TUNEL analyses revealed that in trout vitellogenic follicles, more thecal/epithelial cells than granulosa cells showed fragmented DNA in vivo, but incubation (24 h) did not result in increased apoptosis in cells of either type. These results indicate that apoptosis is involved in normal ovarian growth and postovulatory regression in teleosts, but that it does not appear to be an early event in teleost follicular atresia.