Glyphosate Interference in Follicular Organization in the Wall Lizard Podarcis siculus

Abstract

Glyphosate (Gly) is a broad-spectrum herbicide widely used thanks to its high efficiency and low toxicity. However, evidence exists of its toxic effects on non-target organisms. Among these, the animals inhabiting agricultural fields are particularly threatened. Recent studies demonstrated that exposure to Gly markedly affected the morphophysiology of the liver and testis of the Italian field lizard Podarcis siculus. The present study aimed to investigate the effects of the herbicide on the female reproductive system of this lizard in order to have a full picture of Gly-induced reproductive impairment. The animals were exposed to 0.05 and 0.5 μg/kg of pure Gly by gavage for 3 weeks. The results demonstrated that Gly, at both doses tested, profoundly interfered with ovarian function. It induced germ cells' recruitment and altered follicular anatomy by anticipating apoptotic regression of the pyriform cells. It also induced thecal fibrosis and affected oocyte cytoplasm and zona pellucida organizations. At the functional levels, Gly stimulated the synthesis of estrogen receptors, suggesting a serious endocrine-disrupting effect. Overall, the follicular alterations, combined with those found at the level of the seminiferous tubules in males, suggest serious damage to the reproductive fitness of these non-target organisms, which over time could lead to a decline in survival.

Keywords: estrogen disruptors; oogenesis; reproductive toxicology; reptiles.

Figure 1

Effects of glyphosate (low LD and high HD doses) on follicle anatomy. Significant increase in prefollicular diplotene oocytes (A) and primary follicles (B) (***, p < 0.001). (C) Oocyte with regular cytoplasm (*), zona pellucida (zp), and polymorphic epithelium composed of small (s), pyriform (p), and intermediate (i) cells. Connective theca (t). (D) Apoptotic follicle cells (arrows) in the epithelium (e) and disorganized oocyte cytoplasm (*). (E) Detail of several early apoptotic cells (arrows). Notice the loss of contact with adjacent cells. Group of thecal cells (t). (F) Detail of a patch of condensed follicular epithelium (e). Cells still have a nucleolus (arrow) and the theca is compacted (t). (G) Thickened epithelium (e) with several apoptotic cells (arrow). Altered oocyte cytoplasm (*). (H) Detail of the oocyte cytoplasm with homogeneous (*) and grossly fibrous (arrow) areas. (I) Zona pellucida with discontinuities (arrow). Galgano’s trichrome staining; scale bars: (C–G) 25 µm; (H,I) 10 µm.

Figure 2

Collagen localization in P. siculus follicles exposed to glyphosate (low LD and high HD doses). Picrosirius red staining observed under bright-field light (left panels) or polarized light (right panels). (A,C,E) Progressive thickening and compaction of the connective theca (t). Epithelium (e), zona pellucida (arrows), and oocyte cytoplasm (*). (B,D,F) Progressive increase in mature (red) and immature (green, arrows) fibers in connective theca. Scale bars 20 μm.

Figure 3

(A) Quantitative examination of polarized light images of PSR-stained micrographs. Comparison of mature and immature collagen deposition in high dose (HD) and low dose (LD) vs. control (CTRL) (* p < 0.05; ** p < 0.001); HD vs. LD (# p < 0.001). (B) Mean collagen fiber diameter in HD- and LD-treated samples compared with the control group. HD and LD vs. CTRL (** p < 0.001); HD vs. LD (# p < 0.001).

Figure 4

Effects of glyphosate (low LD and high HD doses) on carbohydrate distribution. PAS staining. (A,B) Stained zona pellucida (zp) and connective theca (t). Faintly stained epithelial cells (e) and oocyte cytoplasm (*). (C) Appearance of stained granules in follicle cells (arrow). Dense theca (t). (D) Release of PAS-positive granules (*) into the oocyte cytoplasm (arrow). Apoptotic cell (*). (E) Dense, poorly stained amorphic bodies (arrows); stained theca (t) and apoptotic pyriforms (*). (F) Disorganized zona pellucida (zp (arrow); PAS-positive granules (arrowhead) in the oocyte cortical cytoplasm. Scale bars: (A) 20 µm; (B–F) 10 µm.

Figure 5

Effects of glyphosate (low LD and high HD doses) on carbohydrate distribution in ovarian follicles. Staining with fluorescent lectins. (A) Stained zona pellucida (arrow) and oocyte cortical granules (*); unstained theca (t) and epithelium (e). (B) Stained zona pellucida (arrows), follicular epithelium (e), and theca (t); unstained oocyte cortical cytoplasm (*). (C) Stained zona pellucida (arrow) and cortical granules (*); unstained epithelium (e). (D,G) Stained theca cells (t), zona pellucida (arrow), and cytoplasm of epithelial cells (e); note the increase in cortical granules (*). Inset: detail of theca with stained cells (arrowhead). (E,H) Unstained theca (t), epithelium (e), and oocyte cytoplasm (*). (F,I) Disappearance of staining on zona pellucida (arrow) and cortical granules (*). WGA stains N-Acetyl-glucosamine, UEA stains fucose, and Con A stains mannose. Scale bars: (A–D; F–I) 20 µm; (E) 40 µm.

Figure 6

Immunohistochemical localization (brown areas) of E-cadherin in follicles of P. siculus treated with glyphosate (low LD and high HD doses). (A,B) Diffuse signal on the zona pellucida and follicular cells. Note the intense signal on theca externa (dotted arrows). (C,D) Intense signal (arrows) on the cytoplasm of occasional pyriform (P) cells and on theca (t). The epithelium (e) in a smaller oocyte is almost unstained and shows apoptotic pyriform cells (*). Zona pellucida (zp) is always stained. (E,F) Signal is on most pyriform (P), intermediate (I), and theca cells (t). E inset: negative control of the reaction. Scale bars: left images, 40 μm; right images, 20 μm.

Figure 7

Immunohistochemical localization (brown areas) of PCNA in follicles of P. siculus treated with low (LD) or high (HD) doses of glyphosate. (A–C) Signal is evident on small (s) and theca cells’ (t) nuclei and on the nuclei of S cells migrating toward the zona pellucida (arrows). Intermediate (i) and pyriform (p) cell nuclei are moderately stained. (D) Intensely stained small (s) and theca (t) cell nuclei. Notice the stain on migrating S cells (arrows) and the presence of early apoptotic pyriform cells (*). (E) Stained small (s), pyriform (p), and theca (t) cell nuclei. Notice the stain on migrating S cells (arrows) and nuclei and/or cytoplasm of intermediate cells (i) (arrowheads). Occasional unstained S cells (dotted arrow) and apoptotic pyriforms (*). (F,G) Stained (arrows and unstained (dotted arrows) small cell nuclei. Notice the stain on migrating S cells (arrows), the occasionally unstained S cells (dotted arrows), and the presence of apoptotic pyriforms (*). Scale bars: 20 µm.

Figure 8

Immunohistochemical localization (brown areas) of ER α and ER β in follicles of P. siculus treated with low (LD) or high (HD) doses of glyphosate. (A,B) Weak signal on the epithelium. Small (s), intermediate (i), pyriform (p), and theca (t) cells. Zona pellucida (zp). (C,D) Appearance of an intense signal on the cytoplasm of occasional intermediate (i) and pyriform (p) cells. (E,F) Intensification of the signal on intermediate (i) and pyriform (p) cell cytoplasm. Stained theca cells (t), unstained small (s) cells, and zona pellucida (zp). Scale bars: 20 µm.