Comparison of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) as test species in the Fish Sexual Development Test (FSDT)

Results are presented from a validation (with 5 laboratories) of the Fish Sexual Development Test (FSDT) developed to detect endocrine disrupters (EDs) and included in the OECD (Organisation for Economic Co-operation and Development) working program. The aromatase-inhibiting fungicide prochloraz was tested in zebrafish (Danio rerio) and fathead minnow (Pimephales promelas). The fish were exposed during sexual differentiation and development from 0 to 60 days post hatch (dph). After exposure, the vitellogenin (VTG) concentrations were quantified in head/tail homogenate and the sex ratio was determined (defined as female, male, intersex or undifferentiated). NOEC/LOEC and EC(x) designs were compared to optimize the test approach. Results show that both species are highly sensitive to prochloraz during sexual development. They respond by skewing of the sex ratio towards male phenotype and by a VTG decline in females. The NOEC/LOEC approach is preferred because sex ratio is difficult to analyze with a regression model. The mean NOEC/LOEC for prochloraz on the sex ratio was 43.3/134 μg/L and 101/293 μg/L for zebrafish and fathead minnow, respectively. The mean NOEC/LOEC on the decline in female VTG concentration was 65/110 μg/L and ~30/68 μg/L respectively. In conclusion, zebrafish and fathead minnow are suitable species in the FSDT and their sexual differentiation is equally labile to EDs.     

Endocrine modulating actions of a phytosterol mixture and its oxidation products in zebrafish (Danio rerio)

In this study, a phytosterol preparation ("ultrasitosterol"; 80% beta-sitosterol) and an oxidized ultrasitosterol preparation were evaluated for reproductive effects in zebrafish. Adults were exposed in a continuous flow to 10 microg/L and 100 microg/L ultrasitosterol and oxidized ultrasitosterol, and to 0.27 microg/L 17beta-estradiol and 0.28 microg/L testosterone for 3 weeks. Biomarkers analysed included plasma vitellogenin, testosterone, 11-ketotestosterone, 17beta-estradiol, and gonadal histopathology. Ovarian steroid production of testosterone and 17beta-estradiol was examined in isolated zebrafish follicles exposed in vitro to the compounds at the same concentrations as in vivo. Vtg was induced in males exposed to ultrasitosterol, and in males and females exposed to 17beta-estradiol. Males exposed to oxidized phytosterols showed increased levels of testosterone and 11-ketotestosterone, and accelerated spermatogenesis. Increased follicular atresia was observed in females exposed to oxidized phytosterols and 17beta-estradiol. Correlation analyses between biomarkers revealed more intercorrelated values for females than for males, and the strongest associations were found in females exposed to oxidized phytosterols. Testosterone production was significantly increased in follicles exposed to the oxidized phytosterol preparations. These findings indicate that the phytosterol mixture is weakly estrogenic in male fish, and that the oxidized phytosterol mixture contains substances that may interfere with spermatogenesis, oogenesis and gonadal steroidogenesis in zebrafish.     

Vitellogenin-immunohistochemistry in the liver and the testis of the medaka, Oryzias latipes, exposed to 17beta-estradiol and p-nonylphenol

Vitellogenin (VTG) produced in male fish has been used for a biomarker to study endocrine disrupters. However, the characteristics of VTG produced in male fish have not been studied well. In this study, we investigated the localization of VTG in the liver and the testis of male medaka (Oryzias latipes) treated with 17beta-estradiol (E2) and p-nonylphenol (NP). The male fish were exposed to 1 microg/L E2 and 500 microg/L NP for 1-12 days. Control groups were kept in water including only vehicle. The frozen sections of the liver and the testis were stained with immunohistochemical methods using an antiserum against medaka VTG as the first antibody. In the E2 and NP treated liver, the hepatocytes showed immunoreactivity. In particular, the cytoplasm close to the cell membrane surrounding the sinusoids was strongly immunopositive. In the testis of both treatments, the interstitial tissues and the cells (spermatocytes) in the seminiferous tubules were immunopositive. The concentration of VTG became gradually higher in both tissues with longer treatments. These results suggest that germ cells in the testis treated with E2 and NP are able to incorporate and accumulate VTG.     

Effects of nonylphenol and phytoestrogen-enriched diet on plasma vitellogenin, steroid hormone, hepatic cytochrome P450 1A, and glutathione-S-transferase values in goldfish (Carassius auratus)

The effects of nonylphenol (NP) on plasma vitellogenin (VTG) and steroid hormone values, as well as hepatic cytochrome P450 1A (CYP1A) and glutathione-S-transferase (GST) activities, were measured in goldfish (Carassius auratus) fed a diet with a low (formulated diet, FD) or high (commercial diet, CD) content of phytoestrogens, including genistein and daidzein. Male goldfish with secondary sexual characteristics were exposed to nominal NP concentrations of 0.1, 1.0, 10, and 100 microg/L in the water for 28 days while being fed either the FD or CD diet at 1.0% of body weight daily. Plasma VTG concentration in male goldfish exposed to 100 microg of NP/L and fed FD was significantly higher than that in the FD-fed control fish at seven, 21, and 28 days. However, fish of the CD-fed group exposed to 100 microg of NP/ L had significantly higher plasma VTG concentration than did fish of the CD-fed control group at 28 days only. Moreover, plasma VTG concentration in fish of the CD-fed control group was about 100-fold higher than that in fish of the FD-fed control group. Although the estrogenic effects of a phytoestrogen-enriched diet caused a decrease in testosterone and/or 11-ketotestosterone values in the CD-fed fish, there was no dose-response relationship between androgen and amount of NP to which the FD-fed fish were exposed. Nonylphenol does not have appreciable effects on hepatic CYP1A and GST activities in male goldfish at concentrations as low as 100 microg/L. These results suggest that NP has estrogenic activity in male goldfish at the nominal concentration of 100 microg/L, and that phytoestrogens, such as genistein and daidzein, in the CD inhibit an aspect(s) of steroid release and/or synthesis common to testosterone and 11-ketotestosterone. However, results of in vivo screening assays for endocrine-disrupting chemicals may be seriously affected by phytoestrogens in the diet, depending on content or potency of estrogenic activity; therefore, we recommend use in research of a standardized, open-formula diet in which estrogenic substances have been reduced to amounts that do not alter the results of studies that are influenced by exogenous estrogens.     

Long term hypoxia suppresses reproductive capacity in the estuarine fish, Fundulus grandis

Human nutrient input has significantly altered dissolved oxygen (DO) cycles in coastal waters such that summertime hypoxia (DO <2 mg/L) and anoxia of bottom water are common worldwide. Prolonged hypoxia usually reduces metabolic rate in fish and potentially reduces reproduction, particularly in a spring and summer spawning species such as the Gulf killifish, Fundulus grandis. To evaluate the effects of long term hypoxia on reproduction, Gulf killifish were subjected to either normoxia (6.68+/-2.1 mg/L DO) or hypoxia (1.34+/-0.45 mg/L DO) for one month. Fecundity, growth, gonadosomatic index (GSI), circulating sex steroids (testosterone, T; 11-ketotestosterone, 11KT; and estradiol-17beta, E2), and egg yolk protein (vitellogenin, VTG) were measured. Hypoxia significantly reduced growth and reproduction. E2 was 50% lower in females and 11KT was 50% lower in males, although the precursor hormone T was unchanged in either sex after hypoxic exposure. Hypoxia-exposed females produced significantly fewer eggs and initiated spawning later than control fish. Plasma VTG concentration was unchanged, suggesting that hypoxia may delay VTG uptake by oocytes. Long term laboratory exposure clearly suppressed reproductive capacity in Gulf killifish. Wild populations experience cyclic hypoxia which could have equivalent effects if daily hypoxic periods are long and frequent - a potential consequence of anthropogenic nutrient enrichment in marsh systems.     

Effects of cadmium on the reproductive axis of Japanese medaka (Oryzias latipes)

Cadmium (Cd) is a ubquitous element and a significant inorganic pollutant that has previously been found to bioaccumulate in reproductive organs of fish and disrupt important endocrine processes, especially those involved in synthesis, release and metabolism of hormones. Clearly, there is potential for reproductive effects in fish populations exposed to Cd, however, few studies have investigated the non-lethal consequences of Cd in fish. To this extent, adult male and female Japanese medaka were exposed to 0-10 ppb Cd for 7 weeks. Reproductive endpoints were monitored during weeks 6 and 7 of exposure and compared to physiological responses along the hypothalamus-pituitary-gonadal (HPG) axis, including plasma vitellogenin (VTG), hepatic estrogen receptor (ER), plasma steroids, gonadal-somatic indices (GSI), and gonadal steroid release. There were no observed effects on VTG and ER by long-term Cd exposure. However, gonadal steroid release was significantly decreased in males and females at all exposure concentrations and female plasma estradiol levels were significantly altered at concentrations higher than 5 ppb Cd. Overall, responses along the HPG axis were more sensitive to Cd exposure than the reproductive and developmental endpoints, which were not affected in this study, indicating that higher level impairment in fish might be relatively protected.     

Effects of nonylphenol on vitellogenin synthesis in adult males of the spotted ray Torpedo marmorata

The aim of this investigation was to assess the effects of nonylphenol (NP), an oestrogen-like environmental pollutant, on the vitellogenin (VTG) synthesis in adult males of the aplacental viviparous cartilaginous fish Torpedo marmorata. The VTG recovery in males is considered a biomarker of xeno-oestrogenic pollution as this lipophosphoglycoprotein is physiologically induced by oestrogens only in females of oviparous and ovoviparous vertebrates. Using in situ hybridization and immunohistochemistry, T. marmorata males injected with nonylphenol showed the presence of VTG in the liver and the kidney. In particular, vtg messenger (m)RNA and VTG protein were expressed in the liver, whereas in the kidney cells only the presence of VTG was recorded. By contrast, no expression for VTG was detected in the testis. These results demonstrate that in T. marmorata NP induces the expression of vtg only in the liver; the presence of VTG in the kidney and its absence in the testis are discussed.     

Effects of pentachlorophenol on the reproduction of Japanese medaka (Oryzias latipes)

Pentachlorophenol (PCP) is widely used to control termites and protect wood from fungal-rot and wood-boring insects, and is often detected in the aquatic environment. Few studies have evaluated PCP as an environmental endocrine disruptor. In the present work, Japanese medaka (Oryzias latipes) was exposed to PCP for 28 days (F0 generation) with subsequent measurements of vitellogenin (VTG), hepatic 7-ethoxyresorufin-O-deethylase (EROD), and reproductive endpoints. Plasma VTG significantly increased in male fish treated with PCP concentrations lower than 200 microg/l and decreased in male and female animals exposed to 200 microg/l. Hepatic EROD from female fish increased when PCP exposure concentrations exceeded 20 microg/l, but decreased in the 200 microg/l PCP treatment group. Fecundity and mean fertility of female medaka decreased significantly in the second and third week following exposure concentrations greater than 100 microg/l, and testis-ova of male medaka was observed at PCP concentrations greater than 50 microg/l. Histological lesions of liver and kidney occurred when exposure concentrations exceeded 50 microg/l. In F1 generations, the hatching rates and time to hatch of offspring were significantly affected in fish exposed to 200 microg/l. These results indicated that PCP exposure caused responses consistent with estrogen and aryl hydrocarbon receptor activation as well as reproductive impairment at environmentally relevant concentrations.     

Influence of parental and developmental cadmium exposure on endocrine and reproductive function in Japanese medaka (Oryzias latipes)

Cadmium (Cd) is a ubiquitous element and an important anthropogenic metal contaminant. A series of assays were modified or developed for Japanese medaka (Oryzias latipes), and used to compare the effects of Cd exposure on indicators of endocrine function in adult animals previously exposed in ovo or as hatchlings. Adults were raised either from eggs produced during a 2 week exposure to 0-10 microg/l Cd or from fry exposed for 2 weeks beginning 2 days after hatching. The reproductive capacity of the resulting adults was determined during a 2 week period during which half of the animals were re-exposed to Cd. Two week Cd exposure did not result in reproductive impairment despite producing some changes in circulating steroid concentration. In addition, 1 microg/l cadmium exposure in ovo elevated male hepatic vitellogenin (VTG) relative to controls. Hence, steroid parameters were a better biomarker of cadmium exposure than changes in VTG. However, reproductive impairment was not correlated to change in VTG or plasma steroids.     

Development and maturation in the nereidid polychaetes Platynereis dumerilii and Nereis succinea exposed to xenoestrogens

Few studies link biochemical, cellular and whole animal effects of toxic compounds with growth and reproductive output on invertebrate model organisms. Thus, we explore the effects of xenoestrogens on nereid worms. Larvae of Platynereis dumerilii exposed to estradiol (E(2)) ethynylestradiol (EE(2)) and nonyplhenol (NP) observing the effects on growth, primordial germ cell (PGC) proliferation and maturation. In addition, a single exposure study was performed with a 50 days latency period on adult worms of Nereis succinea. Since reduced glutathione (GSH) is required in detoxification of NP and is the precursor of the spawning pheromone nereithione (CSSG) in N. succinea, we analysed how the estrogenic chemical NP affects GSH concentrations. PGC were not affected by exposure to E(2) and EE(2) from 24hpf to 6 days. Chronic exposure of P. dumerilii with NP over the full life cycle did not influence segment proliferation. Mature females that developed, even at high concentrations, were able to spawn and successful fertilization occurred. However, at high NP levels no P. dumerilii males matured. A significant decline of GSH can be seen in N. succinea males upon treatment with NP, but not in females, indicating that females stabilize GSH levels even in stress situations. This study shows some results that link the foundation to causally integrate toxic exposure to xenoestrogens with development, growth and reproductive outputs in nereidid polychaetes.     

Reproductive biology and implications for management of the painted sweetlips Diagramma pictum in the southern Arabian Gulf

The reproductive biology of the painted sweetlips Diagramma pictum was determined from 487 individuals collected between January and December 2010 in the southern Arabian Gulf. There was no evidence of sex change and the combination of histological results with the sex composition of the size and age structures indicated a gonochoristic sexual pattern. There were peaks in gonado-somatic indices for females in March and October with spawning occurring during two seasons (April to May and November). The mean size and age at sexual maturity (L(m50) and A(m50) ) were 35·7 cm fork length (L(F) ) and 2·9 years for females and 26·7 cm L(F) and 0·5 years for males. The maximum recorded age (11 years) and small mean size and young age at sexual maturity for males may be a direct result of intensive demersal fishing in the southern Arabian Gulf. There was an exponential increase in the cumulative reproductive potential with size and a linear increase with age for both sexes. The mean L(F) (L(c50) ) at which D. pictum became vulnerable to capture was 33·3 cm, which corresponded to only 3 and 7% of the cumulative reproductive potential of males and females, respectively. Size-specific and age-specific reproductive potential indicated that conventional regulations that equate the mean size at first capture to sexual maturation are unsuitable for the management of D. pictum.     

Sublethal effects of carbaryl on embryonic and gonadal developments of zebrafish Danio rerio

Carbaryl is a broad-spectrum insecticide used to control insect pests. In aquatic environments, it can disrupt the endocrine system and adversely affect the reproductive function of aquatic animals. This study investigated sublethal impacts of carbaryl on embryos and gonads of zebrafish Danio rerio in order to assess the pesticide's impact on its reproduction. Fertilised embryos were exposed to 1.7 mg l^–1 carbaryl until hatching, while larvae aged 10 days post hatching were exposed to 0.1, 0.2, 0.4, 0.8 and 1.7 mg l^–1 carbaryl concentrations until 50 days post hatching (dph). Treatments were applied in a static renewal system and all experiments involved water only and a solvent control. At the end of 50 dph, all surviving fish were sacrificed and processed for light microscopy. Results indicated a mean hatching success rate of 92.5% for control groups, while embryos exposed to carbaryl recorded an 81.0% success rate. Sex reversal was delayed in the experimental groups, with a sex ratio of 13 females to 0 males, but the control group recorded 6 females to 8 males. These results suggest that sublethal doses of carbaryl in the environment, similar to those used in the current study, may have an adverse effect on the reproductive success of zebrafish.     

Correlation between plasma steroid hormones and vitellogenin profiles and lunar periodicity in the female golden rabbitfish, Siganus guttatus (Bloch)

Characteristics of the lunar reproductive cycle in the golden rabbitfish, Siganus guttatus, were determined by histological observations of ovarian development, and immunological measurements of plasma steroid hormones, estradiol-17beta (E2), testosterone (T), 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) and 17alpha,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), and vitellogenin (VTG). Ovarian and plasma samples were collected every week according to the lunar phases from May to July. Weekly change of gonadosomatic index (GSI) showed two peaks at the first lunar quarter in June and July. Yolky oocytes were also observed around this time. Histological observations revealed that the vitellogenic oocytes appeared again 1 week after spawning and developed synchronously. These results suggest that this species is a multiple spawner and the oocyte development is in a group-synchronous manner. Plasma steroid hormones (E2, T, DHP and 20beta-S) and VTG levels changed in parallel with changes in GSI. The peak of plasma VTG level occurred prior to spawning. These cyclic changes of plasma steroid hormones and VTG support the hypothesis that lunar periodicity is the major factor in stimulating reproductive activity of S. guttatus.     

Free female mate choice in house mice affects reproductive success and offspring viability and performance

We tested a critical assumption of sexual dialectics theory (Gowaty 1997, Feminism and Evolutionary Biology, Chapman & Hall) using house mice, Mus musculus. We asked if female house mice accrue viability benefits for their offspring when they mate with males they prefer versus with males they do not prefer. Our experiment was designed to eliminate or control other mechanisms of reproductive competition besides female mate choice. After allowing females to discriminate behaviourally between two males, which were at random with respect to phenotypic variation discriminating females were paired with preferred (P) or nonpreferred (NP) males. We then tested whether females mating with males they preferred had offspring of higher viability than females mating with nonpreferred males. In pairwise comparisons, we tested for differences in offspring performance in dominance contests and in nest-building skill. At weaning, we exposed half of the pups to cold stress. We tested progeny performance and viability in the laboratory or in outdoor field enclosures. In comparison to P females, NP females produced significantly fewer litters. Sons from P matings were socially dominant to sons from NP matings. Adult offspring from P matings built better nests than those from NP matings. In field enclosures significantly fewer NP than P offspring survived to 60 days after introduction. Male and female progeny from P matings established larger home ranges and constructed better nests than progeny from NP matings. This is the first demonstration of progeny viability differences for females allowed to express mate preferences between males presented to them at random. Copyright 2000 The Association for the Study of Animal Behaviour.     

Reproductive biology of Pseudocorynopoma doriai (Pisces: Characidae) in the High Basin of the Samborombón River, province of Buenos Aires, Argentina

The reproductive period of the Glandulocaudine Pseudocorynopoma doriai was determined by the analysis of 240 females, 90 males and 138 immature individuals collected monthly in the Manantiales and the El Portugués rivers. The reproductive period is seasonal, occurring from late winter to mid‐summer, with another reproductive peak of smaller magnitude in early autumn. The mean monthly GSI in males has a significant correlation with rainfall. Other analyzed environmental variables, including temperature, photoperiod, pH and conductivity, did not show a correlation with the mean monthly GSI in females and males. First maturity in females was reached within the 42–43 mm standard length class. Like other Glandulocaudines from southern Brazil, males initiate sexual maturation before females, which implies an adaptive advantage in that this would enable females to spawn under optimal environmental conditions. The mean absolute fecundity was 1286.42 oocytes (SD = 496.9083); the mean relative fecundity was 0.5070 (SD = 0.1333) oocytes by milligram body weight.     

Length–weight and length–length relationships,gonadosomatic indices and size at first maturity of Eugerres mexicanus (Steindachner, 1863) (Percoidei: Gerreidae) from the Usumacinta River,Mexico

This study examines the length‐weight (L‐W) and length‐length (L‐L) relationships, gonadosomatic indices, and size at sexual maturity for individuals of the endemic Mexican mojarra, Eugerres mexicanus (Steindachner, 1863) from the Usumacinta River Basin, Tabasco, Mexico. From April 2008 to January 2010, 360 specimens were examined, including 151 (41.9%) male, 179 (49.7%) female and 30 unsexed (8.3%) individuals. Overall female:male ratio was 1.2 : 1. Gonadosomatic index (GSI) values indicate increased reproductive activity during February, with significant differences between males (GSI = 2.07) and females (GSI = 5.48). The mean size at first maturity (L₅₀) was 17.3 cm TL for males and 20.5 cm TL for females.     

Paternal body size affects reproductive success in laboratory-held zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>)

Across many fish species, large females tend to exhibit higher individual reproductive success due to elevated fecundity and the provisioning of better conditioned eggs and offspring compared to small females. By contrast, effects of paternal body size on reproductive success are less well understood. We disentangled the maternal- and paternal-size dependent effects on reproductive output and early life history in zebrafish (Danio rerio). In the laboratory, females and males from four size categories (small, medium-sized, large and very large) were allowed to spawn freely in a full factorial design with 10 replicates per size combination. As expected, larger females produced more eggs and better conditioned offspring compared to smaller females. Male body size further contributed to zebrafish reproductive success: offspring sired by large males exhibited higher hatching probability and these offspring also hatched earlier and larger than offspring fertilized by small males. However, the largest males experienced lower mating success and received fewer eggs than males of the smaller size classes. While male body size substantially affected reproductive success in zebrafish, it remained unclear whether and to what degree direct paternal effects (e.g., related to sperm quality) or indirect paternal effects stemming from differential allocation patterns by females were the mechanism behind our findings. Answering this question constitutes an important future research topic.     

Reproductive biology of the fanray, <Emphasis Type="Italic">Platyrhina sinensis</Emphasis> (Batoidea: Platyrhinidae) in Ariake Bay,Japan

The reproductive biology of the fanray, Platyrhina sinensis, was examined in Ariake Bay, Japan, from May 2002 until December 2006. Females reached sexual maturity at a larger size than males [total length (TL) at 50% sexual maturity: male, 393 mm; female, 421 mm]. The present data support a distinct annual reproductive cycle for P. sinensis. The gonadosomatic index (GSI) for mature males showed a clear seasonal trend, declining from August to November. Histological observations showed that mature sperm in the testes occurred from August to November when monthly GSI declined. Concomitantly, pre-ovulatory ova were observed in females collected from August to November. These data indicate that mating, ovulation and fertilization occurred from August to November. Near-term embryos, neonates and recent post-partum females also occurred from August to November. Additionally, all post-partum females possessed large pre-ovulatory ova. Parturition occurred from August to November followed immediately by mating, ovulation and fertilization. Mature females become pregnant every year, and the gestation period is almost 1 year. Fertilized uterine eggs without macroscopic embryonic development were present throughout the annual reproductive cycle, indicating that P. sinensis utilizes embryonic diapause as its reproductive strategy. Both reproductive tracts of females were functional, and fecundity ranged from 1 to 12 with a mean of 6.0, increasing with TL.     

Reproductive seasonality of the detritivorous fish <Emphasis Type="Italic">Cyphocharax abramoides</Emphasis> (Kner, 1958) (Characiformes: Curimatidae) in flooded rivers of the eastern Amazon

The Lower Anapu River, Eastern Amazon, has a discrete flood pulse with narrow flooded areas due to the reduced influence of tides. In this environment, local precipitation might act as an important factor in the input of detritus, which can serve as a trigger for fish reproductive period. Therefore, this paper aims to determine the effects of local precipitation on the reproductive ecology of an abundant detritivorous fish in flooded rivers. Between May 2012 and April 2013, 886 specimens (471 females and 415 males) were captured monthly using gill nets. Individuals were measured, weighted, sexed, and the gonadal development stage was defined macroscopically and confirmed histologically. Sex ratio, length-weight relationship (LWR), condition factor (k), Gonadosomatic Index (GSI), relative frequency of maturity stages, and size at first gonadal maturity (L₅₀) were estimated and tested in relation to rainfall periods. There was no difference between the expected sex ratio (1:1), and both males and females showed polyphasic growth. The condition factor indicated higher values at the beginning of the rainy season and lower values during the dry season for both sexes. GSI and relative frequency of mature individuals indicated a long reproduction period with gonadal investment starting within the dry season until early in the rainy season for spawning within the rains. The L₅₀ value showed that males reach sexual maturity before females. Results indicated a reproductive pattern strongly influenced by rainfall, corroborating the importance of local precipitation for lacustrine environments.