Multigenerational and Transgenerational Effects of Environmental Estrogen on Embryonic Development and Reproductive Function in Marine Medaka (Oryzias Melastigma)


Student thesis: Doctoral Thesis

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Award date15 Dec 2022


The synthetic estrogen 17α-ethinylestradiol (EE2) is an endocrine disrupting chemical (EDC) that is widely used in oral contraceptives and hormone therapy. Household waste and urine are the principal routes for EE2 to enter the environment. The chemical characteristics of EE2 led to its persistence in the environment, where it potentially threatens the health of humans and other organisms. Multigenerational studies in mammals and fish have shown that exposure to EE2 impairs reproductive functions such as fecundity, fertilization success, and sperm quality. The cellular and molecular mechanisms of these effects, however, are poorly understood. Additionally, studies of EE2 exposure in embryos suggest that EE2 induces embryo developmental toxicity, including a reduction in embryo survival rates and hatching success. However, the timing and manner of these impairments in the embryo have been less studied, and the mechanisms of EE2-induced embryo developmental toxicity remain to be elucidated.

Moreover, ancestral EE2 exposure (50 ng/L, 7 days) was found to reduce the fertilization rate in F2 and F3 adults and decreased the survival rate of F2 and F3 embryos in Japanese medaka (Oryzias latipes). Additionally, ancestral EE2 exposure may alter gene expression of kisspeptin (kiss1, kiss2) and its receptors (kiss1r and kiss2r), gonadotrophin-releasing hormone (GnRH) and its receptors, and reproduction-related genes such as steroidogenic acute regulatory protein (StAR), the steroidogenic enzyme aromatase Cyp19a1a, and the sex steroid hormone receptors ERα and ERβ. Although previous studies have focused primarily on the direct effects of EE2 exposure, there is increased interest in investigating transgenerational effects. Comparatively fewer transgenerational studies have been conducted, however, so the underlying genetic and molecular mechanisms are not well understood.

In the present study, a multigenerational experiment was conducted to assess the effects of environmental exposure to EE2 on reproductive function and embryo developmental toxicity from F0 to F3, and to decipher the molecular and genetic mechanisms for the multigenerational and transgenerational effects of EE2 exposure. Marine medaka fish (Oryzias melastigma) in the F0 generation were exposed to an environmentally relevant concentration of EE2 at 85 ng/L for 45 days. Their offspring (F1) were divided into two groups for the multigenerational and transgenerational study: one group was continually exposed to 85 ng/L EE2 until the F3 generation, while the other group was transferred to clean artificial seawater until the F3 generation. In each generation, sexually mature adults were assessed for reproductive-related parameters, including Fulton's condition factor, gonadosomatic index, hepatosomatic index, fertilization rate, fecundity, and sperm motility. Embryos from each generation were assessed for developmental toxicity, including developmental delay, developmental rate, heart rate, hatching rate, and larval locomotion.

Our results showed that multigenerational EE2 exposure led to reproductive impairments across generations, including fecundity, fertilization rate, and sperm motility, and induced embryo developmental toxicity in each generation, as evidenced by delayed embryonic development, a reduction in heart rate, decreased hatching success rate and suppression of larval locomotion. Transgenerational EE2 exposure increased the fertilization rate and percentage of motile sperm in the F3 generation, decreased the hatching success, but did not result in significant changes in embryonic development. Moreover, we found that eye pigmentation (both the timing and the percentage of appearance) could serve as a sensitive biomarker in embryo developmental toxicity.

To determine the association between reproductive impairments and embryo developmental toxicity induced by EE2 at the phenotypic and tissue level with the molecular level, an analysis of the transcriptome of the testis and embryo was carried out to determine the relevant genetic pathways. Differentially expressed genes (DEGs) were annotated and enriched with KOBAS-i to identify the altered Gene Ontology (GO) pathways, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, DEGs were enriched with Ingenuity Pathways Analysis (IPA) for altered canonical pathways and gene networks. Our bioinformatics results suggest that multigenerational EE2 exposure may affect androgen synthesis and androgen metabolism, thereby leading to changes in gonadal development and impairments in reproductive fitness. The DEGs of dnm1, pink1, pde7b, and slc12a7 may play an essential role in the reproductive system. In addition, multigenerational EE2 exposure could affect eye development, as well as neurons, and synaptic structure, working in concert to affect the visual and locomotory systems genes such as prkaa2, apob, ephb2, oxtr, nr2e3, and pou4f2 were found to be essential regulators of embryo developmental toxicity. Moreover, ancestral EE2 exposure could alter the pathways related to reproduction and locomotion, such as the retinol, GDNF family ligand receptor and GnRH signaling in the F1 generation.

In summary, these results highlight the biological processes and genes involved in EE2-induced toxicity. Our study provides an ecotoxicological basis for multigenerational and transgenerational EE2 effects on the reproductive system and embryonic development.

    Research areas

  • 17α-ethinylestradiol, reproductive function, embryo developmental toxicity, marine medaka, mRNA network