Environmental Estrogen Induced Multigenerational (F1-F4) Reproductive and Immune Impairments in Fish

Abstract

Estrogenic endocrine disrupting chemicals (EEDCs) occur ubiquitously in aquatic environments and have the potential to impact the endocrine systems in wildlife and humans. Many EEDCs are known to interfere with the endogenous estrogen networks responsible for the regulation of reproduction, growth, metabolism and immune function in vertebrates. Numerous studies have reported the sub-lethal impacts of EEDC exposure on reproductive fitness and, more recently, on immune competence of directly exposed fish. However, the majority of these studies had a single focus on either reproduction or immune function. Reproductive fitness is one of the most important Darwinian fitness traits for determining the sustainability of a population. In addition, the development and maintenance of a healthy immune system is highly energy intensive and important for determining survival fitness. Early life stage mortality, particularly, is high in fish, and the ability of individuals to survive to sexual maturity can directly determine the population size, altering reproductive output and, thus, population sustainability. Surprisingly, a combined reproductive and immune competence assessment of EEDC’s impacts on fish is not available (as well as for other animal models). The findings will definitely be crucial for holistic assessment of the risk of environmental EEDCs on the sustainability of exposed fish populations.

Moreover, growing evidence in mammals and fish indicates that ancestral exposure to EEDCs is able to impact the unexposed offspring in a transgenerational manner ( F2). Inherited epigenetic modifications can only pass to subsequent generations via the germline epigenome of the directly exposed generations. In fish, there is a dearth of information on the dose-response, threshold level, and critical windows (parental gametogenesis and embryogenesis) for assessing the transgenerational risk of EEDC induced reproductive and immune impairments. Furthermore, how fast transgenerational altered phenotypes occur (wash-in) and their persistence through subsequent generations (wash-out) remains unclear. These knowledge gaps are essential for assessing the long-term risk of EEDCs on the future generations of the exposed individuals. Therefore, a multigenerational assessment of the combined reproductive and immune competence after ancestral EE2 exposure is urgently needed for holistic assessment of EEDC/EE2 risk on population sustainability.

In mammals and fish, multigenerational epigenetic modifications in DNA methylation and histone modifications have been shown to be inherited through the paternal and/or maternal germlines. To ascertain how EEDC induced impairments are transgenerationally inherited, the underlying epigenetic and genetic mechanism(s) must be elucidated.

A multigenerational approach was undertaken to assess the multiple impacts of environmental estrogen, 17α-Ethinylestradiol (EE2), on reproduction and immune function of adult Oryzias melastigma (F0) and their offspring (F1-F4). Three different environmentally relevant exposure scenarios were studied, taking into consideration the exposure concentration, duration and critical windows. The exposure regimes used in this experiment were a short parental exposure (SPE), long parental exposure (LPE) and a combined parental and embryonic exposure (PEE) using two environmentally realistic concentrations of EE2: 33 ng/L (Low EE2) and 113 ng/L (High EE2). Reproductive fitness of fish was evaluated by an array of phenotypic endpoints including: fecundity, fertilization rate, hatching success and sex ratio. Immune competence was assessed for larvae (7 days post hatching) and adults through host-resistance assays (HRA) against bacterial challenge and monitoring post-infection fish mortality. To elucidate the potential multigenerational impacts of EE2, the same battery of reproductive and immune endpoints was employed for F1-F4 progeny.

This study is the first of its kind to assess the combined reproductive and immune impacts of EE2 on a transgenerational scale. The identified combined reproductive and immune impacts were assessed in the F1-F4 generations to determine whether the sustainability of the fish populations may be impacted. The results confirm that both parental gametogenesis and embryogenesis are critical windows for EE2 induced transgenerational reproductive impacts. Ancestral exposure to EE2 impaired fertilization success in the F4 LPE High EE2 treatment, and reduced both fertilization success and fecundity in the F4 LPE Low EE2 offspring. The PEE embryonic exposure to High EE2 was able to feminize the directly exposed F1 generation, followed by subsequent masculinization of the F2 and F3 generations, and it returned to a 1:1 ratio (M : F) in the F4 generation. However, the reproductive competence of these F4 offspring was impaired, as indicated by reduced fertilization success, hatching success and delayed hatching time of the F5 offspring. A sex difference was found in the transgenerationally impaired reproductive output, with F4 females being more sensitive than F4 males to a low concentration (33 ng/L) of ancestral parental EE2 exposure (21 days). Conversely, F4 males were more sensitive than F4 females to ancestral embryonic EE2 exposure. For immune competence, no significant transgenerational impacts were identified in the male and female adults. However, seven days of parental gametogenesis exposure (SPE) to 33 ng/L EE2 was found to be a critical window for inducing transgenerational (F3, F4) alterations in the larval survival fitness / immune competence. Overall, ancestral EE2 exposure can likely transgenerationally reduce population sustainability due to impaired reproductive competence in the F4 generation.

Furthermore, the vast amount of experimental data generated from these transgenerational studies are important for expanding the understanding of the temporal dynamics of altered phenotypes over generations (F1-F4). Three dominant transgenerational temporal dynamic models for the appearance and disappearance of significantly altered phenotypes were identified in this study, which occurred most often in the F2 (Moderate-in) and F3/F4 (Slow-in) generations, and the altered phenotypes usually disappeared after a single generation (Fast-out). The findings provide strong evidence on the wash-out behavior of altered phenotypes over generations. The importance of assessing phenotype prevalence in a population in order to gain a deeper understanding of the “non-significant” presence of altered phenotypes is discussed. These novel findings provide important insights into the multigenerational temporal patterns of altered phenotypes, their implications on population sustainability and their potential underlying genetic and epigenetic mechanisms.

To elucidate the underlying genetic and epigenetic mechanism(s), the transgenerational impaired fertilization success and hatching success in the F4 PEE treatment was further assessed at the tissue/cellular level (testis histopathology and sperm motility) and biochemical level (plasma sex hormones 11-KT, T and E2) in order to uncover possible changes in spermatogenesis. RT-QPCR was used to identify perturbations of key genes involved in the major spermatogenesis related pathways in testis and sperms. Epigenetic modifications in global DNA methylation and genome wide DNA methylation as well as histone methylation (H3K4me2 and H3K27me2) were investigated in testis and sperm to provide pilot data on the potential epigenetic marks and male germline transmission of altered phenotype initiated by ancestral EE2 exposure. In the F4 High EE2 males reduced sperm motility, altered testis morphology, and reduced abundance of spermatogonia, spermatocytes and spermatids were found, indicating impaired spermatogenesis. The male sex steroid 11-KT, a primary regulator of spermatogenesis, was significantly decreased. In addition, the female oocyte promoting gene FOXL2 was found to be significantly unregulated, indicating disruption of the downstream spermatogonial germ cell proliferation pathways. Of the 281 differentially methylated genes identified in the High EE2 F4 sperm, SOX8, a suppressor of FOXL2 was found hypermethylated at the promoter and gene body. Evidence of the underlying genetic and epigenetic modifications in DNA methylation and histone H3K4me2 in the testis and sperm leading to the F4 transgenerational altered reproductive phenotypes are discussed. In summary, these pilot findings suggest that EE2 is a transgenerational reproductive toxicant that can alter the male germ line epigenome leading to impaired spermatogenesis in the F4 generation.

Overall, these novel results highlight the importance of a concomitant assessment of reproductive and immune competence after ancestral EEDCs exposure for at least four generations. This comprehensive study provides a toxicological basis for multigenerational risk assessment associated with EEDCs in the aquatic environments. The findings of EE2 induced transgenerational impairment in fish also sheds light on the potential multigenerational impacts of a single exposure event in other vertebrates.

Date of Award	17 Jan 2018
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Wai Ting Doris AU (Supervisor)