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. 2023 Nov;160(5):453-475.
doi: 10.1007/s00418-023-02222-4. Epub 2023 Jul 26.

Zinc-nanoparticles alleviate the ovarian damage induced by bacterial lipopolysaccharide (LPS) in pregnant rats and their fetuses

Abd El-Fattah B M El-Beltagy  1 , Samaa M Bakr  2 , Samah S G Mekhaimer  2 , Noura F Ghanem  2 , Amany Attaallah  3
Affiliations

Zinc-nanoparticles alleviate the ovarian damage induced by bacterial lipopolysaccharide (LPS) in pregnant rats and their fetuses

Abd El-Fattah B M El-Beltagy et al. Histochem Cell Biol. 2023 Nov.

Abstract

Lipopolysaccharide (LPS) is an endotoxin derived from the cell wall of Gram-negative bacteria. LPS exposure during early gestation is associated with adverse effects on the placenta as well as on developmental outcomes, including embryonic resorption, fetal death, congenital teratogenesis, and fetal growth retardation. This work aimed to explore the adverse effects of LPS injected at an early stage of gestation on the gonads of pregnant rats and the ovaries of their pups and the role of zinc nanoparticles (Zn-NPs) against these adverse effects. Twenty-four pregnant rats were used in this study. They were divided at gestation day 4 into four groups (n = 6): control, Zn-NPs (20 mg/kg orally from gestation day E14 till the end of weaning), LPS (50 µg/kg at gestation days E7 and E9), and LPS + Zn-NPs group. The body weight and placenta weight were recorded at gestational day 16. At postnatal day 21 (weaning), the mothers rats and their offspring were sacrificed and immediately dissected to remove the ovaries and uteri from the mothers and the ovaries from their offspring for subsequent biochemical, histological, and immunohistochemical investigations. The obtained results revealed that LPS exposure during early gestation caused severe histopathological alterations in the placenta, uterus, and ovaries of mothers, as well as in the ovaries of their pups. Also, the uterine and ovarian sections displayed a positive reaction for caspase-3 antibody and a negative reaction for Bcl-2 antibody, which reflects the apoptotic effect of LPS. Additionally, remarkable reductions in the levels of antioxidants (superoxide dismutase and catalase) and significant increases in malondialdehyde (MDA) levels were recorded in the serum of LPS-treated mothers and in the ovarian tissues of their offspring. Further biochemical analysis of the ovarian tissues from LPS-maternally treated offspring showed a significant increase in the levels of caspase-3, TNF-α, and TGF-β1, but a significant decrease in the level of IGF-1. On the other hand, treatment of mothers with Zn-NPs from day 14 of gestation until the weaning day (21st day postnatal) successfully ameliorated most of the deleterious histopathological, immunohistochemical, and biochemical changes induced by LPS.

Keywords: Antioxidants; Apoptosis; Gestation; LPS; Ovary; Pups; Zn-NPs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
The body weight and placenta (not uterine) weight changes in pregnant rats at 16th day of gestation (panels a and b), and body weight changes at PND21 (panel c). Data was expressed in mean ± SD. P value for the comparison between the four studied groups P1: P value for comparing between LPS and LPS + Zn-NP, *Statistically significant at P ≤ 0.05. #Significant with control, @significant LPS with LPS + Zn-NP group in graph
Fig. 2
Fig. 2
Photomicrograph of histological sections through the ovaries among the different studied groups of mother rats (a control, b Zn-NP, c LPS, and d LPS + Zn-NP). The ovarian sections from control and Zn-NP-supplemented pregnant rats appeared to have normal histological architecture. In LPS-treated rats, the ovarian section showed atretic follicles (AF), dispersed pyknotic cells (PyC), and degenerated stroma (DS) with obvious congested blood vessels (CBV). In the LPS group cosupplemented with Zn-NP, the ovarian stroma, as well as the follicles, appear intact, while the little follicles appear atretic (stain H&E, scale bar 25 µm). A antrum, TF theca folliculi, GC granulosa cells, O oocyte, CL corpus luteum, S stroma, DS degenerated stroma, PrF primordial follicles, PF primary follicles, AF atretic follicles, PyC pyknotic cells, and CBV congested blood vessels
Fig. 3
Fig. 3
Photomicrograph of histological sections through the uterus among the different studied groups of mother rats (a control, b Zn-NP, c LPS, and d LPS + Zn-NP). The uterine sections from control and Zn-NP-supplemented rats appear with normal histological architecture. In LPS-treated rats, the uterine section showed congested capillaries (white asterisks) in the myometrium, as well as fragmented myometrial muscle fibers (FMM), fibrotic tissue in endometrium (arrows), and fragmented endometrial epithelium (FEE). In LPS group cosupplemented with Zn-NP, the uterine layers appear well organized (stain: H&E, scale bar: 100 µm). MM myometrium, UE uterine endometrium, EE endometrial epithelium, EG endometrial glands, L lumen, FMM fragmented myometrium, FEE fragmented endometrial epithelium
Fig. 4
Fig. 4
Photomicrograph of histological sections through the placenta among the different studied groups of mother rats at the 16th day of gestation (a control, b Zn-NP, c LPS, and d LPS + Zn-NP). The placenta sections from control and Zn-NP-supplemented rats appear to have normal histological architecture. In LPS-treated rats, the placenta layers appear disorganized, with congested capillaries (blue asterisk) and fibrotic tissues (green asterisk). Stain: H & E, scale bar: 25 µm. MG metrial glands, De decidua, BZ basal zone (trophospongium), LZ labyrinth zone
Fig. 5
Fig. 5
Photomicrograph of paraffin-embedded sections through the ovaries of the different studied groups of female rats stained with Bcl-2 antibody (a control, b Zn-NP, c LPS, and d LPS + Zn-NP). Panels (a and b) display weak to moderate Bcl-2 expression in the follicular cells and little area in the ovarian stroma. In the LPS-treated group (panel c), the degree of Bcl-2 immunoreactivity appears negative in the cells of mature follicles and very weak in the ovarian stroma. In LPS-treated rats cosupplemented with Zn-NP (panel d), the ovarian stroma shows weak immune expression for Bcl-2, while the follicular cells moderate Bcl-2 expression (Bcl-2 antibody, scale bar: 25 µm). The arrow heads refer to the immunoreactivity of Bcl-2
Fig. 6
Fig. 6
Image analysis for Fig. 5 showing the % of Bcl-2 positively stained area of the ovarian sections among the different studied groups of mother rats
Fig. 7
Fig. 7
Photomicrograph of paraffin-embedded sections through the uterine endometrium of the different studied groups of female rats stained with Bcl-2 antibody (a control, b Zn-NP, c LPS, and d LPS + Zn-NP). The endometrial glands appear moderately stained with Bcl-2 antibody in control and Zn-NP-treated group (panels a and b), negatively stained in the LPS-treated group (panel c), and weakly stained in the LPS + Zn-NP group (panel d). Bcl-2 antibody, scale bar: 100 µm. The arrow heads refer to the immunoreactivity of Bcl-2
Fig. 8
Fig. 8
Image analysis for Fig. 7 showing the % of Bcl-2 positively stained area of the uterine sections among the different studied groups of mother rats
Fig. 9
Fig. 9
Photomicrograph of paraffin-embedded sections through the ovaries of the different studied groups of female rats stained with caspase-3 antibody (a control, b Zn-NP, c LPS, and d LPS + Zn-NP). The follicular granulosa cells and ovarian stroma appear weakly stained with caspase-3 antibody in control and Zn-NP (panels a and b), strongly stained in the LPS-treated group (panel c), and weakly stained in the stroma but negatively stained granulosa cells in the LPS + Zn-NP group (panel d). Caspase-3 antibody, scale bar: 25 µm. The arrow heads refer to the immunoreactivity of caspase-3
Fig. 10
Fig. 10
Image analysis for Fig. 9 showing the % of caspase-3 positively stained area of the ovarian sections among the different studied groups of mother rats
Fig. 11
Fig. 11
Photomicrograph of paraffin-embedded sections through the uterine endometrium of the different studied groups of female rats stained with caspase-3 antibody (a control, b Zn-NP, c LPS, and d LPS + Zn-NP). The endometrial glands appear weakly stained with caspase-3 antibody in control and Zn-NP (panels a and b), strongly stained in the LPS-treated group (panel c), and moderately stained in the LPS + Zn-NpP group (panel d). Caspase-3 antibody, scale bar: 100 µm. The arrow heads refer to the immunoreactivity of caspase-3
Fig. 12
Fig. 12
Image analysis for Fig. 11 showing the % of caspase-3 positively stained area of the uterine sections among the different studied groups of mother rats
Fig. 13
Fig. 13
Shows the levels of serum CAT (a), SOD and MDA (b), and FSH (c) among the different studied groups of mother rats
Fig. 14
Fig. 14
Photomicrograph of histological sections through the ovaries among the different studied groups of 21-day-old offspring (a control, b Zn-NP, c LPS, and d LPS + Zn-NP). In the control and Zn-NP groups, the ovarian sections appear to have a well-organized pattern of ovarian follicles embedded in the ovarian stroma. In the LPS group, the ovarian follicles appear to be few, and most of them are atretic. In LPS + Zn-NP, the ovarian follicles show remarkable amelioration in their architecture. Stain: H&E, scale bar: 25 µm. A antrum, GC granulosa cells, O oocyte, TF theca folliculi, S stroma, AF atretic follicles, * ruptured follicles
Fig. 15
Fig. 15
Photomicrograph of paraffin-embedded sections through the ovaries of the different studied groups of 21-day-old offspring stained with Bcl-2 antibody (a control, b Zn-NP, c LPS, and d LPS + Zn-NP). In control and Zn-NP (panels a and b), the ovarian sections display moderate Bcl-2 expression in the follicular cells. In the LPS-treated group (panel c), the degree of Bcl-2 immunoreactivity appears very weak in the cells of some follicles. In LPS-treated rats cosupplemented with Zn-NP (panel d), the follicular cells show moderate Bcl-2 expression. In all groups, the ovarian stroma appeared negatively stained for Bcl-2 protein (Bcl-2 antibody, scale bar: 25 µm).The arrow heads refer to the immunoreactivity of Bcl-2
Fig. 16
Fig. 16
Image analysis for Fig. 15 showing the % of Bcl-2positivelly stained area of the ovarian sections among the different studied groups of 21 day old offspring
Fig. 17
Fig. 17
Photomicrograph of paraffin-embedded sections through the ovaries of the different studied groups of 21-day-old offspring stained with caspase-3 antibody (a control, b Zn-NP, c LPS, and d LPS + Zn-NP). In control and Zn-NP groups (panels a and b), the immunoreactivity for caspase-3 appears very weak in the follicular cells and negative in the ovarian stroma. In the LPS-treated group (panel c), the degree of caspase-3 immunoreactivity appears strongly expressed in both follicular cells and the ovarian stroma. In LPS-treated rats cosupplemented with Zn-NP (panel d), the caspase-3 activity appears very weak in the follicular cells and negative in the ovarian stroma. Caspase-3 antibody, scale bar: 25 µm. The arrow heads refer to the immunoreactivity of caspase-3
Fig. 18
Fig. 18
Image analysis for Fig. 17 showing the % of caspase-3 positively stained area of the ovarian sections among the different studied groups of 21-day-old offspring
Fig. 19
Fig. 19
Changes in the levels of serum SOD, CAT (panel a), MDA (panel b), caspase-3 (panel c), TNF-α, TGF-β1 (panel d), and IGF-1 (panel e) among the different studied groups of 21-day-old offspring
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