Influences of DMP on the fertilization process and subsequent embryogenesis of abalone (Haliotis diversicolor supertexta) by gametes exposure

Abstract

Di-methyl phthalate (DMP), a typical endocrine disrupting chemical (EDC), is ubiquitously distributed in aquatic environments; yet studies regarding its impact on gametes and the resulting effects on embryogenesis in marine gastropods are relatively scarce. In this study, the influences of DMP on the gametes and subsequent developmental process of abalone (Haliotis diversicolor supertexta, a representative marine benthic gastropod) were assessed. Newborn abalone eggs and sperm were exposed separately to different DMP concentrations (1, 10 or 100 ppb) for 60 min. At the end-point of exposure, the DMP-treated eggs and sperm were collected for analysis of their ultra-structures, ATPase activities and total lipid levels, and the fertilized gametes (embryos) were collected to monitor related reproductive parameters (fertilization rate, abnormal development rate and hatching success rate). Treatment with DMP did not significantly alter the structure or total lipid content of eggs at any of the doses tested. Hatching failures and morphological abnormalities were only observed with the highest dose of DMP (100 ppb). However, DMP exposure did suppress sperm ATPase activities and affect the morphological character of their mitochondria. DMP-treated sperm exhibited dose-dependent decreases in fertilization efficiency, morphogenesis and hatchability. Relatively obvious toxicological effects were observed when both sperm and eggs were exposed to DMP. Furthermore, RT-PCR results indicate that treatment of gametes with DMP changed the expression patterns of physiologically-regulated genes (cyp3a, 17β-HSD-11 and 17β-HSD-12) in subsequent embryogenesis. Taken together, this study proofed that pre-fertilization exposure of abalone eggs, sperm or both to DMP adversely affects the fertilization process and subsequent embryogenesis.

Figure 1. Effects of DMP exposure on total lipid levels of eggs and ATPase activities of sperm.

(A) total lipid levels of eggs. (B) ATPase activities of sperm. Each bar represents the mean ± SD. Data are representative of three independent experiments. Significant differences (P<0.05, one-way ANOVA) in total lipid levels and ATPase activities between the experimental and control groups are indicated with different letter.

Figure 2. Light microscope images of an abalone sperm (A) and egg (B).

The full-length of the sperm is approximately 45 µm, and the diameter of the egg is approximately 150 µm.

Figure 3. Electron microscopic analysis of the effect of DMP treatment on abalone sperm and eggs.

A, the ultrastructure of control sperm (the arrows indicate mitochondria, the insert picture is the full TEM image of the sperm); B, the ultrastructure of DMP-treated sperm (the mitochondria swell and secund cristae are marked by dotted line arrows); C, the ultrastructure of control eggs; and D, the ultrastructure of DMP-treated eggs (arrows denote egg envelopes).

Figure 4. Effects of DMP exposure on fertilization rates (%) of abalone gametes.

The percentages of fertilization in different protocols were determined by counting approximately 100–150 randomly sampled eggs. Data are means±SD of three tests. Different letters denote statistically significant differences between control and treatment groups determined by one-way ANOVA (^bP<0.05, ^cP<0.01).

Figure 5. Effect of PAEs on morphological development of abalone embryos.

Representative malformations in abalone embryos due to exposure of gametes to DMP (taking sperm exposed to 10 ppb DMP as a example). Arrows point to representative abnormalities for each stage, including incomplete cleavage (1), abnormal protuberances on the embryo envelope (2), dissymmetry splits (3, 4), yolk-sac edema (5, 6) and developmental arrest (7) or monotrochal larva malformation (8).

Figure 6. Effects of DMP exposure on embryo abnormality rates (%) of abalone gametes.

Values are means±SD, and different letters denote values that are significantly different (P<0.05) and the columns that share the same letter are not significantly different (P>0.05).

Figure 7. Effects of DMP exposure on Embryo hatching rates (%) of abalone gametes.

Values are means±SD, and different letters denote values that are significantly different (P<0.05) and the columns that share the same letter are not significantly different (P>0.05).

Figure 8. Effect of DMP on reference genes expression in abalone embryos.

Expression levels of cyp3a, 17β-HSD-11 and 17β-HSD-12 after exposure of sperm, eggs or both to DMP. Each bar represents the mean±SD. All real-time reactions were performed three times. Significant differences among the groups were indicated with different letter.

Figure 9. The experimental animals in this study.

A, the fully-mature abalone (shell-length near 7 cm); B, the male individual (note the yellowish-white spermary); and C, the female individual (note the dark-violet ovary).