Ovarian regression and apoptosis in the South American teleost Leporinus taeniatus Lütken (Characiformes, Anostomidae) from the São Francisco Basin

Involution and resorption of both postovulatory and atretic follicles were analysed in piau‐jejo Leporinus taeniatus (Characiformes, Anostomidae) in order to evaluate the role of apoptosis during ovarian regression. Histological and ultrastructural analyses showed hallmarks of apoptosis in the granulosa: aggregation of compacted chromatin against the nuclear envelope, cell shrinkage, surface blebbing, loss of cell adhesion and cell fragmentation into apoptotic bodies. Protein synthesis activity preceded the onset of the cell death. The breakdown of the basement membrane led to the detachment of the granulosa cells into the follicular lumen. TUNEL‐positive reactions were detected in in situ DNA fragmentation of granulosa of both postovulatory and atretic follicles. Apoptosis increased in a time‐dependent manner contributing to reduction of the follicular areas. The apoptotic index (per cent of apoptotic cells) of the granulosa increased in postovulatory follicles soon after spawning, then these follicles degenerated and only remnants were observed at 7 days. In contrast, the granulosa cells reabsorbed the yolk during follicular atresia and the apoptotic index increased only in the late stage of regression. The results indicated apoptosis as the major mechanism to rapidly eliminate postovulatory follicles and being an essential process in the ovarian regression after spawning.     

Ovarian follicular atresia is mediated by heterophagy, autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes)

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.     

Ovarian follicular atresia is mediated by heterophagy,autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes)

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.     

Postovulatory follicle: a model for experimental studies of programmed cell death or apoptosis in teleosts

This is the first evidence of programmed cell death, or apoptosis, occurring in the postovulatory follicle (POF) of teleost fish. Females of Astyanax bimaculatus lacustris were submitted to induced ovulation through injecting pituitary extract. Ultrastructural analyses of POFs at time intervals varying from zero to four days postspawning showed several characteristic events of the apoptosis. Typical apoptotic figures, such as nucleus with chromatin condensation underlying the nuclear envelope in a crescent pattern and apoptotic bodies at different stages of formation and reabsorption, were observed in the follicular cells a few days after the onset of the postovulatory period. The results indicated that apoptosis is the major mechanism responsible for the elimination of the follicular cells in the POFs of A. bimaculatus lacustris during ovarian recovery postspawning. It is suggested that POFs might be used as an experimental model in dynamic studies involving cell death in teleosts.     

Dual roles for autophagy during follicular atresia in fish ovary

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., non-ovulated) 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 vacuoles containing degenerating organelles increasing through follicular atresia in both species. In this addendum, we propose a hypothesis for follicular cell removal during ovarian regression in oviparous fish. In this model, autophagy could have dual roles in follicular atresia. Thus, fish ovaries after breeding are suitable models for studying the interactions among the different cell death pathways.     

Patterns of resorption of vitellogenous oocytes in the striped flounder Pleuronectes pinnifasciatus

Two patterns of resorption of vitellogenous oocytes with the participation of follicular cells, differing in their morphological features, were revealed in the striped flounder Pleuronectes pinnifasciatus. In the prespawning season, the resorption affected a small number of oocytes with yolk granules and resembled in some features (destruction of the nucleus and radial coating) the atresia of ovarian follicles in many fish species previously studied. In the spawning season, resorption was specified by accretion of the follicular epithelium and release of the homogenized yolk from the oocytes to the follicular wall. No destruction of the radial coating was observed in that case, and the trophic material was utilized in protrusions of the follicular wall.     

Reproductive Timing of Fishes in a Tropical Intermittent Stream

Six fish species were studied in an intermittent lowland stream in Trinidad, West Indies for 28 months from 1980 to 1982. They were Gasteropelecus sternicla, Corynopoma riisei, Astyanax bimaculatus, Hemigrammus unilineatus, Corydoras aeneus and Poecilia reticulata. Peaks in GSIs and presence of mature adults and juveniles, as determined from monthly field collections, indicated reproduction coincident with the rainy seasons in all species except Poecilia. Gasteropelecus bred for a short period at the beginning of the main rainy season while Corynopoma, Corydoras and Hemigrammus had extended periods of breeding activity each year coincident with high rainfall. The presence of juveniles suggests that Astyanax also bred during the rainy season. Poecilia reproduced continuously throughout the study period but exhibited discharge-related adult stock fluctuations that may have influenced reproductive activity. Maximum ovarian fecundity correlated with species size suggested body size limitation on fecundity for small species such as Corynopoma and Hemigrammus. Size frequency distributions of oocytes in mature ovaries indicate synchronised ovum maturation in Astyanax and possibly Gasteropelecus, while Hemigrammus, Corynopoma and Corydoras showed evidence of continuous maturation of oocytes and multiple spawning. Prolonged small brood spawning was confirmed in the laboratory for Corynopoma and Hemigrammus and resulted in spawning fecundities up to 11 times greater than ovarian fecundities for Corynopoma. It is concluded that the conditions of intermittency in this stream did not significantly alter the reproductive styles of these species as described for other Neotropical environments, perhaps because of their opportunistic nature. Patterns of spawning for the characoids can be directly related to size and phylogeny.     

The relationship between stomach contents and maturity state for major northwest Atlantic fishes: new paradigms?

Iteroparous species examined from the northwest Atlantic demonstrated distinct relationships between feeding and reproduction. Two species showed an increase in feeding during the developing, ripe and running stages of reproduction, indicative of the ramper pattern. Four species showed an increase in feeding after spawning in the spent or resting stage of reproduction, indicative of the rester pattern. Two species exhibited no distinct pattern. Evidence from other species confirms these major patterns. This information may be useful to determine the effectiveness of different management measures, particularly in relation to feeding or spawning aggregations. There are two major feeding-reproductive patterns for fishes along a continuum of possibilities.     

Unilateral ovariectomy increases egg size and reduces follicular atresia in the semelparous coho salmon, Oncorhynchus kisutch

Unilateral ovariectomy (ULO, removal of one ovary) is a powerful technique for studying aspects of reproductive physiology, including follicular recruitment and growth. To examine effects of ULO for the first time in a semelparous species, coho salmon (Oncorhynchus kisutch) were unilaterally ovariectomized during mid-vitellogenesis approximately 3 months before spawning. At termination of the study (79 days post-surgery), single ovaries of ULO fish were gravimetrically equivalent to paired ovaries of sham surgery, control fish. There was no evidence of recruitment of new vitellogenic follicles. Instead, the dramatic increase in ovary mass was attributable to hypertrophy of existing vitellogenic follicles (33% increase in volume) and increased fecundity achieved through a greater than two-fold reduction in follicular atresia. The composition of whole ovaries on a dry weight basis from ULO fish was greater in protein, but lower in lipid than that of control fish. Expressing the data on a per follicle basis, however, showed that follicles of ULO fish contained more protein, ash, water, and lipid. The results indicate that ULO of coho salmon induces compensatory hypertrophy of existing vitellogenic follicles, while maximizing fecundity through reduction of atresia. Thus, 3 months before spawning, coho salmon exhibit the ability to adjust final egg size and number when faced with significant depletion of ovarian follicles. This in vivo system provides a platform for further study of physiological mechanisms regulating follicular growth and atresia, and the trade-off between egg size and egg number. J. Exp. Zool. 309A:468-476, 2008. (c) 2008 Wiley-Liss, Inc.     

Collapse of the reproductive process of two migratory fish (Prochilodus argenteus and Prochilodus costatus) in the Três Marias Reservoir,São Francisco River,Brazil

Generally, migratory fish can complete vitellogenesis even when the migration process is interrupted; however, final oocyte maturation does not occur and therefore there is no spawning. To evaluate the reproductive process of migratory fish inside the reservoir of the Três Marias Dam (a lentic environment), 90 female and 81 male Prochilodus argenteus and 172 female and 115 male Prochilodus costatus were captured in this environment. Males and females from both species in the resting stage were pre‐dominant, and the highest frequency of specimens in advanced maturation stages occurred in November, December and January. According to histological analysis, ovarian regression was frequent and vitellogenic oocytes undergoing atresia were intensely observed, moreover, no fish with ovaries and testes with characteristics of spawning and spermiation, respectively, were captured. This indicated that both species were preparing for reproduction and reaching advanced gonad maturation, but were unable to complete the process of reproduction in this environment. The data from this study indicate that the modification of portions of rivers to lentic environments can have a negative impact on the reproductive process of migratory fish in freshwater.     

Expression of hyaluronan synthase 1 and distribution of hyaluronan during follicular atresia in pig ovaries

CD44 on macrophages is recognized as a phagocytic receptor involved in the phagocytosis of apoptotic cells. Recently, we detected CD44 on macrophages in atretic follicles during atresia. In this study, we evaluated the distribution of the principal CD44 ligand hyaluronan (HA) and the expressions of HA synthases (HAS: HAS1, HAS2, and HAS3) during atresia in pig ovaries. We determined the 2139-bp sequence of Sus scrofa HAS1 and raised an anti-HAS1 polyclonal antibody. The S. scrofa HAS1 sequence contained six putative HA-binding motifs and conserved amino acid residues crucial for GlcNac transferase activity. HAS1 mRNA expression was upregulated during atresia; however, HAS2 and HAS3 mRNA expression levels were low and very low to undetectable, respectively. Western blotting showed that HAS1 was markedly upregulated during atresia. Immunohistochemical analyses revealed HAS1 distribution in theca cells of healthy and early atretic (stages I and II) follicles and in progressing atretic (stage III) follicles. Hyaluronan was visualized with the HA-binding protein; it accumulated in the theca layer during all stages and in stage III follicles. Hyaluronan assay showed a significantly increased HA concentration in follicular fluid at stage III. Flow cytometry showed HAS1 expression in 55.7% of SIRPA-positive macrophages in stage III follicles. Our results suggest that the HA concentration in follicular fluids increased during atresia and that HAS1 may be the dominant HAS protein in theca cells to produce HA in pig ovaries.     

Autophagy and apoptosis interplay during follicular atresia in fish ovary: a morphological and immunocytochemical study

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.     

Reproduction of the lambari Astyanax scabripinnis (Jenyns, 1842) (Pisces: Characidae) in a small stream in Southeastern Brazil

The reproduction of Astyanax scabripinnis captured bimonthly in the Cristais small stream (20^o00'03'S and 43^o56'46'W), in the Das Velhas River basin, was studied using gross anatomical and histological techniques. Standard length of females (56 ± 8 mm) was longer than males (49 ± 6 mm). The diameter of the nucleus of the primary spermatogonia was 7.5 ± 0.9  μ m and of the spermatozoon head 1.8 ± 0.4  μ m. For the females, the oogonia nuclei measured 10.2 ± 2.2  μ m and the vitellogenic oocyte nuclei measured 675.0 ± 66.7  μ m. The following stages of the reproductive cycle were established: developing, mature, partially spent/spawned and completely spent/spawned. Maximum number of males (61.3%) and females (58.2%) at the mature stage was observed during November/December. The gonadosomatic index of males and females was highest during the mature stage (2.4 ± 0.6; 7.5 ± 1.3, respectively) and decreased in subsequent stages. In females, the hepatosomatic index was highest during the developing stage (2.8 ± 0.3), and slightly decreased in the following stages. The highest stomach repletion index found for males was observed during the partially spent stage (2.0 ± 1.2), while for the females, this index varied discretely between the reproductive stages. The highest values of Fulton's condition factor were registered during the mature stage in both sexes (2.9 ± 0.4 for males; 3.0 ± 0.3 for females). The continuous reproductive period and the presence of partially spawned females during the entire sampling period indicated that the species has fractional spawning.     

Siderastrea siderea spawning and oocyte resorption at high latitude

Abstract

At high latitudes (>25°), sexual reproduction and the maintenance of coral populations can be impaired by marginal environmental conditions. However, little is known about sexual reproduction of many coral species at high latitude on the northern-most extension of the Florida Reef Tract. This study aimed to histologically characterize the reproductive ecology of Siderastrea siderea, near Fort Lauderdale, Florida (26°N). Tissue samples of S. siderea were collected semi-monthly to multi-weekly from August to November in 2007 and 2008. Spawning was inferred from gametogenesis and oocyte resorption was observed in detail. Environmental variables including temperature and lunar cycle were examined for relationship with potential spawning times. Based on the histological evidence, we infer that spawning likely occurred primarily in October. Gametogenesis in this species is likely mediated by seasonal temperature variation, whereas lunar cycle could act as finer scale environmental cue for coordination of spawning. Our findings highlight that S. siderea spawning occurs later in the year compared to other populations of this species throughout the Caribbean and to other coral species near Fort Lauderdale. For the first time, oocyte resorption stages are described and constitute a baseline for future projects that aim to understand this process in corals.     

Structural characteristics of gonadal development and hermaphroditic phenomenon in freshwater pearl mussel,Sinohyriopisis schlegelii (Hyriopisis schlegelii)

The period of gonads development was first studied from one to five years in the freshwater pearl mussel Hyriopisis schlegelii. It lasted for 36 months and was divided into three main stages: initiation of gonad formation, a stable growth phase, and a reproductive cell development phase. Each reproductive cycle consisted of five stages: proliferative stage (from late January to late February), growth stage (from late February to late March), maturation stage, spawning stage (from early April to late October) and recovery stage (from early November to late January). Interestingly, a hermaphroditic phenomenon was observed in this mussel for the first time, which appears during the development stage from 26 to 32 months. Male and female follicular tissues coexisted in hermaphrodite individuals with the male follicular tissue accounting for more than 90% of the whole gonad tissue. No hermaphroditic phenomenon was observed in matured gonad. We thus speculate that self-fertilization does not exist in H. schlegelii.     

Fecundity regulation by atresia in turbot Scophthalmus maximus in the Baltic Sea

Down‐regulation of fecundity through oocyte resorption was assessed in Baltic Sea turbot Scophthalmus maximus at three locations in the period from late vitellogenesis in April to spawning during June to July. The mean ± s.d . total length of the sampled fish was 32·7 ± 3·1 cm and mean ± s.d . age was 6·2 ± 1·5 years. Measurements of atresia were performed using the ‘profile method’ with the intensity of atresia adjusted according to the ‘dissector method’ (10·6% adjustment; coefficient of determination was 0·675 between methods). Both prevalence (portion of fish with atresia) and intensity (calculated as the average proportion of atretic cells in fish displaying atresia) of atresia were low in prespawning fish, but high from onset of spawning throughout the spawning period. Atretic oocytes categorized as in early alpha and in late alpha state occurred irrespective of maturity stage from late prespawning individuals up to late spawning fish, showing that oocytes may become atretic throughout the spawning period. Observed prevalence of atresia throughout the spawning period was almost 40% with an intensity of c. 20%. This indicates extensive down‐regulation, i.e. considerably lower realized (number of eggs spawned) v. potential fecundity (number of developing oocytes), suggesting significant variability in reproductive potential. The extent of fecundity regulation in relation to fish condition (Fulton's condition factor) is discussed, suggesting an association between levels of atresia and fish condition.     

To breed or not to breed: Physiological correlates of reproductive status in a facultatively biennial iguanid

It is unusual for seasonal breeders to frequently skip opportunities for reproduction. We investigated the relationship between physiological state and reproductive decision-making in Galápagos marine iguanas (Amblyrhynchus cristatus), a species in which females typically reproduce biennially, although the proportion of breeding individuals varies significantly across years. Nearly all adult-sized females initiated follicular development prior to the lekking period, but 38% of females resorbed all developing follicles 5-15 days before the start of copulations. Receptive and non-receptive females differed in reproductive hormones during the mate choice period. Testosterone peaked in receptive females immediately prior to copulation, indicating that testosterone or its derivative estradiol likely mediates female receptivity in Galápagos marine iguanas. Non-receptive females showed significant peaks in both testosterone and progesterone during follicular atresia, suggesting that these hormones may be involved in inhibiting vitellogenesis. Two to three weeks prior to the period of reproductive decision-making (and the onset of follicular atresia in non-receptive females) receptive females were in higher body condition, were developing larger follicles, and had lower levels of both baseline and stress-induced corticosterone. Reproduction is extremely costly in this long-lived species, and increases the likelihood of mortality in the year following breeding; females could therefore gain significant benefits from being attuned to indicators of reproductive success. We suggest that corticosterone may modulate reproductive decisions by altering individual sensitivity to both internal and external cues of the likelihood of successful reproduction.     

Profile of micronucleus frequencies and DNA damage in different species of fish in a eutrophic tropical lake

Lake Paranoá is a tropical reservoir for the City of Brasilia, which became eutrophic due to inadequate sewage treatment associated with intensive population growth. At present, two wastewater treatment plants are capable of processing up to 95% of the domestic sewage, thereby successfully reducing eutrophization. We evaluated both genotoxic and cytotoxic parameters in several fish species (Geophagus brasiliensis, Cichla temensis, Hoplias malabaricus, Astyanax bimaculatus lacustres, Oreochromis niloticus, Cyprinus carpio and Steindachnerina insculpita) by using the micronucleus (MN) test, the comet assay and nuclear abnormality assessment in peripheral erythrocytes. The highest frequencies of MN were found in Cichla temensis and Hoplias malabaricus, which were statistically significant when compared to the other species. However, Steindachnerina insculpita (a detritivorous and lake-floor feeder species) showed the highest index of DNA damage in the comet assay, followed by C. temensis (piscivorous). Nuclear abnormalities, such as binucleated, blebbed, lobed and notched cells, were used as evidence of cytotoxicity. Oreochromis niloticus followed by Hoplias malaricus, ominivorous/detritivotous and piscivorous species, respectively, presented the highest frequency of nuclear abnormalities, especially notched cells, while the herbivorous Astyanax bimaculatus lacustres showed the lowest frequency compared to the other species studied. Thus, for biomonitoring aquatic genotoxins under field conditions, the food web should also be considered.     

Reproductive patterns in seventeen species of warmwater fishes in a Missouri River reservoir

Synopsis The timing of ovarian maturation and spawning of 17 warmwater fish species in Lake Oahe (South and North Dakota) was estimated from changes in the mean ovary indices (ratios of ovary weight to fish length). The onset of vitellogenesis varied within species (up to 2 months). Maturation of the ova took from 7.5 to 10 months, depending on species. Annual variations in the mean date of peak spawning of individual species during 1964–1971 were usually less than a week. There was little overlap of the annual mean peak spawning dates of the 17 species, and an established sequence of spawning among species was shown. A relatively high incidence of atresia in the shovelnose sturgeon, northern pike, and carp indicated that these species had apparently not yet adapted to the altered and variable spawning conditions in this reservoir. Regularity of spawning would seem to provide the best chance for spawning success in variable environments such as Lake Oahe.