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Int J Mol Sci
2024 Feb 06;254:. doi: 10.3390/ijms25041969.
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Benzodiazepine Interference with Fertility and Embryo Development: A Preliminary Survey in the Sea Urchin Paracentrotus lividus.
Fogliano C
,
Carotenuto R
,
Cirino P
,
Panzuto R
,
Ciaravolo M
,
Simoniello P
,
Sgariglia I
,
Motta CM
,
Avallone B
.
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Psychotropic drugs and benzodiazepines are nowadays among the primary substances of abuse. This results in a large and constant release into aquatic environments where they have potentially harmful effects on non-target organisms and, eventually, human health. In the last decades, evidence has been collected on the possible interference of benzodiazepines with reproductive processes, but data are few and incomplete. In this study, the possible negative influence of delorazepam on fertilization and embryo development has been tested in Paracentrotus lividus, a key model organism in studies of reproduction and embryonic development. Sperm, eggs, or fertilized eggs have been exposed to delorazepam at three concentrations: 1 μg/L (environmentally realistic), 5 μg/L, and 10 μg/L. Results indicate that delorazepam reduces the fertilizing capacity of male and female gametes and interferes with fertilization and embryo development. Exposure causes anatomical anomalies in plutei, accelerates/delays development, and alters the presence and distribution of glycoconjugates such as N-Acetyl-glucosamine, α-linked fucose, and α-linked mannose in both morulae and plutei. These results should attract attention to the reproductive fitness of aquatic species exposed to benzodiazepines and pave the way for further investigation of the effects they may exert on human fertility. The presence of benzodiazepines in the aquatic environment raises concerns about the reproductive well-being of aquatic species. Additionally, it prompts worries regarding potential impacts on human fertility due to the excessive use of anxiolytics.
Figure 1. Effects of DLZ on fertilization percentage in the sea urchin Paracentrotus lividus. (A) Pre-treated eggs. (B) Pre-treated sperm. (C) Treatment at fertilization. Significant effects are registered exclusively at the higher dose. (**, p < 0.01; ***, p < 0.001).
Figure 2. Effects of DLZ on cleavage progression: percentage of morulae six hours post fertilization (hpf). (A) Pre-treated eggs. (B) Pre-treated sperm. (C) Treatment at fertilization. (D) Treatment after fertilization. Significant effects are registered only at the higher dose, in (A–C). In sample (D), DLZ significantly delays cleavage at all tested concentrations. (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001 with respect to control).
Figure 3. Effect of DLZ on the composition of the population of plutei. (A) 4-arm plutei represent about two-thirds of the individuals. The remaining one-third is composed of two-arm plutei and occasional morulae. (B–E) Significant variation in population composition. (*, p < 0.5; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 with respect to control).
Figure 4. Effects of DLZ on larval morphology. (a) Morula with dissociated blastomeres (*) in a fertilization membrane (dotted arrows). (b) Abnormally developed morula (*). Fertilization membrane (dotted arrow). (c) Plutei with enlarged tip of the anterior arm (arrows). Pre- (small arrow) and post- (dotted arrow) oral arms. (d) Pluteus with bent arm (arrow). Inset: intact pluteus. (e,f) Detail of apical arms with crossed or split skeletal rods (arrows). (g) Deformed pluteus. (h) Percentages of anomalous plutei after the different experimental treatments. (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001 with respect to control).
Figure 5. Effects of DLZ on gut development. (A) Pluteus with intact gut. Animal length (l) and gut width (w) were determined and used to identify possible anomalies in gut development. (B) Ratio (expressed as w/l × 100), in control samples. (C–F) Ratios after treatments. Notice moderate effects after gamete pre-treatments (C,D), more significant effects after exposure at fertilization (E), and the absence of effects after exposure during development (F). (*, p < 0.05; **, p < 0.01 with respect to control.)
Figure 6. Effects of DLZ on carbohydrate presence and distribution in the morulae (6 hpf). Staining with FITC-lectins; blastomeres (*), fertilization membranes (arrows). (a) Morula with stained blastomeres and fertilization membrane. (b) Unstained blastomeres and stained fertilization membrane. (c) Stained blastomeres and fertilization membrane. (d) Moderately stained blastomeres and stained fertilization membrane. (e) Unstained morula. (f–h) Treatment-dependent staining of blastomeres and fertilization membranes. (i,j) Unstained morulae. WGA stains terminal N-acetyl glucosamine, UEA-I stains α-linked fucose, Con A stains α-linked mannose, DBA and SBA stain α- or β-linked N-Acetyl galactosamine. Bars: 25 µm.
Figure 7. Effects of DLZ on carbohydrate presence and distribution in the plutei (48 hpf). Staining with FITC-WGA (a–f); skeletal rods (dotted arrows), mesenchyme cells (thick arrow), apical mesenchyme cells (thin arrow). (a) Pluteus with stained rods and mesenchyme cells. (b) Poorly stained rods and mesenchyme cells. (c) Intensely stained rods and mesenchyme cells in an abnormal 2-arm pluteus. (d) Unstained pluteus. (e,f) Stained skeletal rods and mesenchyme cells. Staining with FITC-Con A; stomodeum (*), gut (arrow). (g,h,k,m,n) Stained stomodeum and unstained gut. (i,j,l) Unstained stomodeum. WGA stains terminal N-Acetyl glucosamine and Con A stains α-linked mannose. Bars: 25 µm.
Figure 8. Schematic representation of the four treatments carried out. (A,B) Eggs and sperm pre-treatment with DLZ; washing and use in fertilization with untreated sperm or eggs. Development in pure seawater. (C) Fertilization of untreated gametes in the presence of DLZ; washing after the elevation of the fertilization membrane and development in pure seawater. (D) Fertilization of untreated gametes in pure seawater; addition of DLZ after the elevation of the fertilization membrane and development in the presence of DLZ.
