Title & Abstract

International Journal of Anatomy and Research

Protective Effects of Vitamins A, C, and E on Ovarian Histopathology, Apoptotic Signaling, Oxidative Stress, and Early Embryonic Development in Zinc Oxide Nanoparticle-Induced Toxicity in Female Rats

DOI: 10.16965/ijar.2026.178

Al-Rikabi, Sally Adnan Mousa ¹

ABSTRACT

Background.

Zinc oxide nanoparticles (ZnO-NPs) are one of the most common metal oxide nanomaterials produced in the world and their uses are increasing in cosmetics, food packaging, sunscreens and in pharmaceuticals as well as agrochemicals. There is increasing concern on their possible reproductive toxicity, especially on ovarian tissue in females.

Aims.

To investigate the role of oral co-delivery of vitamins A, C, and E (alone and in combination) in reducing ZnO-NP-induced ovarian oxidative damage, histopathological destruction, apoptotic activation, hormonal perturbation, and impaired early embryonic development in adult female Wistar rats.

Methods.

Forty-eight adult female Wistar rats (10-12 weeks, 180-220 g) were randomized into six equal groups (n=8) and treated by oral gavage for 28 days: G1 (saline control), G2 (ZnO-NP 100 mg/kg), G3-G5 (ZnO-NP + vitamin A [1000 IU/kg], C [200 mg/kg], or E [100 mg/kg]), and G6 (ZnO-NP + combined A+C+E). On the 5th gestational day (GD 5) after mating, animals were sacrificed. Assays comprised ovarian oxidative stress biomarkers (MDA, GSH, SOD, CAT), serum reproductive hormones (FSH, LH, estradiol, progesterone), haematoxylin and eosin (H&E) histopathology with semi-quantitative lesion scoring, immunohistochemistry (IHC) of Bax, Bcl-2, caspase-3, TUNEL assay, and blastomere/implantation metrics.

Results.

ZnO-NP exposure resulted in a high level of lipid peroxidation, antioxidant depletion, follicular atresia, vascular congestion, stromal edema, inflammatory infiltration, a 61-fold increase in ovarian Bax/Bcl-2 ratio (0.10 to 6.14), elevated caspase-3 activity, high TUNEL index (67.8%), and reduced blastomere cleavage/implantation. All the vitamins partially reversed these changes; the A+C+E combination produced the overall most protection with all parameters returning to statistically equal levels of controls.

Conclusion.

ZnO-NPs cause multi-level reproductive toxicity by activating the intrinsic apoptotic pathway by oxidative stress in granulosa cells. Combinations of antioxidant vitamin supplements have comprehensive, synergistic effects and should be the subject of additional translational research.

Keywords: zinc oxide nanoparticles; ovarian toxicity; histopathology; apoptosis; Bax; Bcl-2; caspase-3; TUNEL; oxidative stress; vitamin A; vitamin C; vitamin E; follicular atresia; early embryonic development; female rats

INTRODUCTION

Engineered nanomaterials have found their way into numerous industries in the contemporary world and zinc oxide nanoparticles (ZnO-NPs) are some of the most widely manufactured metal oxide nanomaterials on earth. These possess photocatalytic, UV-absorbing, and antimicrobial properties, along with a favourable biocompatibility profile, which has led to their use in sunscreens, cosmetics, food packaging, biomedical imaging agent formulations, feed additives, and agrochemical formulations [1,2]. This extensive use has simultaneously raised significant concerns about unintended biological impacts after occupational, environmental or dietary exposure.

After oral intake, ZnO-NPs partially dissolve in the acidic gastric environment, releasing free Zn2+ ions, and remaining unabsorbed nanoparticles can cross the intestinal epithelial cell via endocytic and paracellular routes. The resultant systemic distribution leads to accumulation in the most highly perfused, metabolically active organs such as the liver, kidneys, spleen, and the gonads [3-5]. Experimental evidence is on the increase that both the particulate and ionic fraction are capable of causing oxidative stress, mitochondrial dysfunction, genotoxicity and apoptosis in a variety of cell types [6,7].

The mammalian ovary is especially susceptible to oxidative attacks since folliculogenesis, steroidogenesis, and meiotic maturation require redox signalling that is highly controlled. The overabundance of reactive oxygen species (ROS) interferes with granulosa cell proliferation, quality of oocytes, follicular atresia, and the hypothalamic-pituitary-gonadal (HPG) axis [8-10]. It has been proven by experimental reports that subchronic exposure to ZnO-NPs increases malondialdehyde (MDA), reduces reduced glutathione (GSH), inhibits superoxide dismutase (SOD) and catalase (CAT) as well as causes morphological damage to ovarian follicles [11,12].

At the heart of the pathophysiology of oxidative follicular damage is the activation of the apoptotic cascade in granulosa cells. The balance between pro-apoptotic Bcl-2 family proteins (specifically, Bax) and anti-apoptotic ones (specifically, Bcl-2) regulates the intrinsic (mitochondrial) apoptotic pathway. Bax facilitates mitochondrial outer membrane permeabilization (MOMP), the release of cytochrome c, which triggers caspase-9 and, subsequently, the executioner caspase-3, resulting in internucleosomal DNA fragmentation, which can be detected using the TUNEL assay under excessive ROS [13-17]; the ratio of Bax/Bcl-2 expression in ovarian granulosa cells is thus a decisive mechanistic index of the propensity to apoptosis in toxic stress.

Biologically valid possibilities to prevent this redox-mediated damage include classical antioxidant vitamins that mitigate the damage at several pathway levels. Vitamin A (retinol) and its metabolites play a role in stabilizing membranes and cell differentiation of follicular and luteal cells through nuclear retinoic acid receptors [18]. Vitamin C (ascorbic acid) is a strong aqueous phase scavenger of peroxyl and hydroxyl radicals, revitalizes oxidized vitamin E and is enriched in follicular fluid [19-21]. Vitamin E (alpha-tocopherol) selectively breaks lipid-peroxidation-chain reactions in biological membranes [22]. Although there is significant attention on the protective effects of these vitamins individually, limited comparative and combined efficacy data against nanoparticle-induced ovarian injury in particular in terms of the apoptotic signalling axis are available. The current study thus aimed to investigate whether in a well-controlled rat model of oral ZnO-NP exposure, supplementation with vitamins A, C, or E, individually and in combination, could prevent ovarian oxidative damage, histopathological destruction, apoptotic activation, endocrine disruption, and early embryonic impairment.

MATERIALS AND METHODS

Chemicals and Nanoparticle Characterization

ZnO-NPs were obtained as uncoated powder (purity: 99%, primary particle size 30 ± 5 nm; Sigma-Aldrich, St. Louis, MO, USA). Particle morphology was confirmed by transmission electron microscopy (TEM; JEOL JEM-2100), dynamic light scattering (DLS; Malvern Zetasizer Nano ZS) for hydrodynamic diameter (95 ± 12 nm), polydispersity index (PDI = 0.18), and zeta potential (-18 ± 2.4 mV), and X-ray diffraction (XRD; Shimadzu XRD-6100) for crystalline phase. Before dosing, suspensions were prepared freshly in sterile 0.9% saline, sonicated (100 W, 40 kHz, 30 min, ice bath), and vortexed. Retinyl palmitate (vitamin A), L-ascorbic acid (vitamin C), and DL-a-tocopheryl acetate (vitamin E) were purchased from Merck (Darmstadt, Germany).

Animals, Experimental Design, and Dosing

This study was carried out at the Kut Technical Institute, Middle Technical University, Wasit Province, Iraq (Ethical Approval No. WU-2125, College of Science, University of Wasit). Forty-eight sexually mature virgin female Wistar rats (10-12 weeks, 180-220 g) and 24 fertile males were maintained under controlled environmental conditions (22°C, 55% relative humidity, 12 h light/dark cycle) with ad libitum standard diet and water following a 7-day acclimatisation period. Rats with regular 4-5 day estrous cycles were randomized into six equal groups (n = 8) and dosed daily via oral gavage for 28 days: G1 (Control, sterile saline 1 mL/kg), G2 (ZnO-NP 100 mg/kg), G3 (ZnO-NP 100 mg/kg + vitamin A [retinyl palmitate 1000 IU/kg]), G4 (ZnO-NP 100 mg/kg + vitamin C [L-ascorbic acid 200 mg/kg]), G5 (ZnO-NP 100 mg/kg + vitamin E [DL-a-tocopheryl acetate 100 mg/kg]), and G6 (ZnO-NP 100 mg/kg + combined vitamins A [1000 IU/kg] + C [200 mg/kg] + E [100 mg/kg]) administered as two individual gavages 30 minutes apart (Table 1).

Table 1

Experimental groups, designations, and daily oral treatments (n = 8 per group).

Group | Label | Daily Treatment (oral gavage, 28 days) | n
G1 | Control | Sterile saline, 1 mL/kg | 8
G2 | ZnO-NP | ZnO-NP, 100 mg/kg | 8
G3 | ZnO-NP + Vit A | ZnO-NP (100 mg/kg) + retinyl palmitate (1000 IU/kg) | 8
G4 | ZnO-NP + Vit C | ZnO-NP (100 mg/kg) + L-ascorbic acid (200 mg/kg) | 8
G5 | ZnO-NP + Vit E | ZnO-NP (100 mg/kg) + DL-a-tocopheryl acetate (100 mg/kg) | 8
G6 | ZnO-NP + A+C+E | ZnO-NP (100 mg/kg) + vitamins A (1000 IU/kg) + C (200 mg/kg) + E (100 mg/kg) | 8

ZnO-NP, zinc oxide nanoparticle; IU, international units.

Mating Protocol and Preimplantation Embryo Collection

Following 28-day treatment, females were cohoused with fertile males overnight (2:1). Gestational day 0 (GD 0) was confirmed by vaginal plug. On GD 5, animals were euthanized (sodium pentobarbital 100 mg/kg i.p.). Blood was collected via cardiac puncture for serum separation. Both ovaries were excised, trimmed, and weighed (right snap-frozen at -80°C for biochemistry; left fixed in 10% neutral buffered formalin for histology). Uterine horns were flushed with 0.4% PBS-BSA to recover and morphologically evaluate preimplantation embryos under stereomicroscopy (x40).

Oxidative Stress, Hormonal Assays, Histopathology, and Apoptotic Profiling

Ovarian homogenates were assayed for MDA (TBARS method), GSH (Ellman's method), SOD (pyrogallol autoxidation), CAT (H2O2 decomposition at 240 nm), and total protein (Lowry method). Serum FSH, LH, 17beta-estradiol (E2), and progesterone (P4) were quantified via validated rat ELISA kits. Paraffin sections (5 µm) were stained with H&E for differential follicle counting and semi-quantitative scoring (0-4 scale: vascular congestion, stromal edema, follicular degeneration, apoptotic bodies, inflammatory infiltration; weighted kappa = 0.88). Immunohistochemistry was conducted for Bax (1:200, Abcam ab32503), Bcl-2 (1:100, Abcam ab182858), and cleaved caspase-3 using DAB chromogen and H-score/percent positive quantification. In situ apoptotic fragmentation was quantified via DeadEnd Fluorometric TUNEL assay (Promega G3250), and caspase-3 enzymatic activity measured via colorimetric Ac-DEVD-pNA cleavage (Abcam ab39401).

Statistical Analysis

Data are presented as Mean ± SEM (n = 8). Normality was verified by Shapiro-Wilk and homogeneity of variance by Levene's test. Group comparisons were conducted using One-Way ANOVA with Tukey's post-hoc HSD test (significance set at p < 0.05) using IBM SPSS v26 and GraphPad Prism v9.

RESULTS

No mortality occurred. Body weight gain was significantly reduced in G2 (27.7 ± 3.1 g vs. 54.4 ± 2.3 g in G1, p < 0.05), alongside absolute ovarian weight reduction (48.3 ± 2.9 mg vs. 71.8 ± 3.2 mg, p < 0.05). Vitamin co-administration progressively restored both metrics, with G6 producing complete recovery (Table 2).

Ovarian MDA nearly tripled in G2 (6.82 ± 0.58 nmol/mg vs. 2.35 ± 0.22 in G1, p < 0.001), while GSH, SOD, and CAT activities fell by 59-61% (p < 0.001). G6 restored MDA to 2.78 ± 0.24 nmol/mg and returned antioxidant enzymes to 85-93% of control values (Table 3). Endocrine evaluation revealed a hypergonadotropic-hypogonadal profile in G2 (elevated FSH/LH, suppressed E2/P4), which was normalized in G6 (Table 4). Follicular reserves were severely depleted in G2 with a 4-fold increase in atresia (p < 0.001), completely ameliorated in G6 (Table 5).

Histopathological scoring (Table 6, Figure 1) showed severe congestion, stromal edema, follicular degeneration, apoptotic bodies, and inflammatory infiltration in G2 (total score 17.1 ± 0.6 vs. 1.4 ± 0.3 in G1, p < 0.05), which was reversed in G6 (3.3 ± 0.3). Ovarian Bax expression rose from 8.2% to 72.5% in G2 while Bcl-2 dropped from 78.4% to 11.8%, elevating the Bax/Bcl-2 ratio 61-fold (6.14 ± 0.42 vs. 0.10 ± 0.01 in G1, p < 0.001) alongside a 5.2-fold elevation in caspase-3 activity (42.5 ± 3.2 pmol/min/mg) and TUNEL index (67.8 ± 4.1%). G6 normalized Bax/Bcl-2 to 0.20 ± 0.02 and TUNEL index to 10.3 ± 0.9% (Table 7, Figures 2 and 3). Implantation rate was severely reduced in G2 (42.7 ± 4.2% vs. 86.4 ± 3.8% in G1, p < 0.001), restored to 80.3 ± 3.7% in G6 (Table 8).

Figure 1

Table 2

Body weight parameters and absolute ovarian weight across experimental groups (mean ± SEM, n = 8).

Group | Initial BW (g) | Final BW (g) | BW Gain (g) | Ovarian Wt (mg)
G1 Control | 194.2 ± 4.1 | 248.6 ± 5.8 | 54.4 ± 2.3 | 71.8 ± 3.2
G2 ZnO-NP | 192.8 ± 3.6 | 220.5 ± 6.4* | 27.7 ± 3.1* | 48.3 ± 2.9*
G3 + Vit A | 193.6 ± 4.3 | 234.1 ± 5.6 | 40.5 ± 2.7+ | 58.4 ± 2.7+
G4 + Vit C | 195.1 ± 3.8 | 236.9 ± 5.2 | 41.8 ± 2.8+ | 60.1 ± 2.5+
G5 + Vit E | 193.0 ± 4.0 | 239.7 ± 5.9 | 46.7 ± 2.6+ | 63.6 ± 2.8+
G6 + A+C+E | 194.7 ± 3.9 | 245.3 ± 5.7+ | 50.6 ± 2.4+ | 68.9 ± 3.0+‡

*p < 0.05 vs. G1 (Control); +p < 0.05 vs. G2 (ZnO-NP); ‡p < 0.05 vs. single-vitamin groups (G3-G5).

Table 3

Ovarian tissue oxidative stress markers and antioxidant enzyme activities (mean ± SEM, n = 8).

Group | MDA (nmol/mg) | GSH (nmol/mg) | SOD (U/mg) | CAT (U/mg)
G1 Control | 2.35 ± 0.22 | 28.6 ± 2.1 | 17.8 ± 1.4 | 42.5 ± 3.1
G2 ZnO-NP | 6.82 ± 0.58* | 11.2 ± 1.3* | 6.9 ± 0.9* | 17.6 ± 2.2*
G3 + Vit A | 4.05 ± 0.34+ | 19.4 ± 1.8+ | 11.8 ± 1.1+ | 28.4 ± 2.6+
G4 + Vit C | 3.88 ± 0.31+ | 20.8 ± 1.9+ | 12.6 ± 1.2+ | 30.2 ± 2.7+
G5 + Vit E | 3.42 ± 0.28+ | 22.7 ± 2.0+ | 13.9 ± 1.3+ | 33.8 ± 2.9+
G6 + A+C+E | 2.78 ± 0.24+‡ | 26.5 ± 2.1+‡ | 16.4 ± 1.4+‡ | 39.7 ± 3.0+‡

MDA, malondialdehyde; GSH, reduced glutathione; SOD, superoxide dismutase; CAT, catalase. *p < 0.001 vs. G1 (Control); +p < 0.05 vs. G2 (ZnO-NP); ‡p < 0.05 vs. single-vitamin groups.

Table 4

Serum reproductive hormone concentrations across experimental groups (mean ± SEM, n = 8).

Group | FSH (mIU/mL) | LH (mIU/mL) | Estradiol (pg/mL) | Progesterone (ng/mL)
G1 Control | 6.8 ± 0.5 | 4.2 ± 0.4 | 48.7 ± 3.2 | 22.4 ± 1.8
G2 ZnO-NP | 11.4 ± 0.7* | 7.9 ± 0.6* | 22.6 ± 2.1* | 10.8 ± 1.2*
G3 + Vit A | 8.9 ± 0.6+ | 5.8 ± 0.5+ | 33.8 ± 2.7+ | 15.9 ± 1.5+
G4 + Vit C | 8.7 ± 0.6+ | 5.6 ± 0.5+ | 35.2 ± 2.8+ | 16.5 ± 1.5+
G5 + Vit E | 8.2 ± 0.6+ | 5.4 ± 0.4+ | 37.5 ± 2.9+ | 17.8 ± 1.6+
G6 + A+C+E | 7.3 ± 0.5+‡ | 4.7 ± 0.4+‡ | 44.9 ± 3.1+‡ | 20.7 ± 1.7+‡

FSH, follicle-stimulating hormone; LH, luteinizing hormone. *p < 0.01 vs. G1; +p < 0.05 vs. G2; ‡p < 0.05 vs. G3-G5.

Table 5

Mean ovarian follicle counts per representative histological section (mean ± SEM, n = 8).

Group | Primordial | Primary | Secondary | Antral | Atretic
G1 Control | 52.3 ± 3.4 | 28.5 ± 2.1 | 18.7 ± 1.5 | 12.4 ± 1.1 | 4.2 ± 0.5
G2 ZnO-NP | 24.8 ± 2.2* | 12.4 ± 1.3* | 7.1 ± 0.9* | 3.8 ± 0.6* | 16.9 ± 1.4*
G3 + Vit A | 36.7 ± 2.6+ | 19.8 ± 1.7+ | 12.9 ± 1.2+ | 7.6 ± 0.8+ | 10.4 ± 1.0+
G4 + Vit C | 37.9 ± 2.7+ | 20.6 ± 1.7+ | 13.4 ± 1.2+ | 7.9 ± 0.9+ | 9.8 ± 1.0+
G5 + Vit E | 40.5 ± 2.9+ | 22.8 ± 1.8+ | 14.8 ± 1.3+ | 9.1 ± 0.9+ | 8.3 ± 0.9+
G6 + A+C+E | 48.1 ± 3.2+‡ | 26.3 ± 2.0+‡ | 17.2 ± 1.4+‡ | 11.0 ± 1.0+‡ | 5.4 ± 0.6+‡

*p < 0.05 vs. G1 (Control); +p < 0.05 vs. G2 (ZnO-NP); ‡p < 0.05 vs. G3-G5.

Table 6

Semi-quantitative histopathological lesion scores (0-4 scale, mean ± SEM, n = 8; inter-observer kappa = 0.88).

Group | Vascular Congestion | Stromal Edema | Follicular Degeneration | Apoptotic Bodies | Inflammatory Infiltration | Total Score
G1 Control | 0.3 ± 0.1 | 0.2 ± 0.1 | 0.4 ± 0.1 | 0.3 ± 0.1 | 0.2 ± 0.1 | 1.4 ± 0.3
G2 ZnO-NP | 3.5 ± 0.2* | 3.3 ± 0.2* | 3.8 ± 0.2* | 3.6 ± 0.2* | 2.9 ± 0.2* | 17.1 ± 0.6*
G3 ZnO-NP + Vit A | 2.3 ± 0.2+ | 2.0 ± 0.2+ | 2.5 ± 0.2+ | 2.4 ± 0.2+ | 1.9 ± 0.2+ | 11.1 ± 0.5+
G4 ZnO-NP + Vit C | 2.1 ± 0.2+ | 1.9 ± 0.2+ | 2.3 ± 0.2+ | 2.2 ± 0.2+ | 1.8 ± 0.2+ | 10.3 ± 0.5+
G5 ZnO-NP + Vit E | 1.8 ± 0.2+ | 1.7 ± 0.2+ | 2.0 ± 0.2+ | 1.9 ± 0.2+ | 1.6 ± 0.2+ | 9.0 ± 0.4+
G6 ZnO-NP + A+C+E | 0.7 ± 0.1‡ | 0.6 ± 0.1‡ | 0.8 ± 0.1‡ | 0.7 ± 0.1‡ | 0.5 ± 0.1‡ | 3.3 ± 0.3‡

*p < 0.05 vs. G1 (Control); +p < 0.05 vs. G2 (ZnO-NP); ‡p < 0.05 vs. single-vitamin groups.

Table 7

Immunohistochemical apoptotic markers and caspase-3 enzymatic activity (mean ± SEM, n = 8).

Group | Bax (+%) | Bcl-2 (+%) | Bax/Bcl-2 Ratio | Caspase-3 (pmol/min/mg) | TUNEL Index (%)
G1 Control | 8.2 ± 1.1 | 78.4 ± 3.2 | 0.10 ± 0.01 | 8.2 ± 0.8 | 5.2 ± 0.7
G2 ZnO-NP | 72.5 ± 3.8* | 11.8 ± 1.5* | 6.14 ± 0.42* | 42.5 ± 3.2* | 67.8 ± 4.1*
G3 + Vit A | 48.3 ± 2.9+ | 38.2 ± 2.8+ | 1.26 ± 0.12+ | 26.8 ± 2.1+ | 41.5 ± 3.0+
G4 + Vit C | 45.1 ± 2.7+ | 41.9 ± 2.9+ | 1.08 ± 0.10+ | 24.5 ± 2.0+ | 38.2 ± 2.8+
G5 + Vit E | 38.4 ± 2.5+ | 50.3 ± 3.1+ | 0.76 ± 0.08+ | 20.3 ± 1.8+ | 31.6 ± 2.5+
G6 + A+C+E | 14.2 ± 1.5+‡ | 72.6 ± 3.2+‡ | 0.20 ± 0.02+‡ | 11.4 ± 0.9+‡ | 10.3 ± 0.9+‡

*p < 0.001 vs. G1 (Control); +p < 0.05 vs. G2 (ZnO-NP); ‡p < 0.05 vs. single-vitamin groups (G3-G5).

Table 8

Early embryonic development parameters at gestational day 5 (mean ± SEM, n = 8).

Group | Implantation Rate (%) | Normal Embryos (%) | Mean Blastomere Count (GD 5)
G1 Control | 86.4 ± 3.8 | 91.2 ± 3.1 | 7.8 ± 0.4
G2 ZnO-NP | 42.7 ± 4.2* | 48.6 ± 3.6* | 3.6 ± 0.3*
G3 + Vit A | 63.8 ± 4.0+ | 68.4 ± 3.4+ | 5.4 ± 0.4+
G4 + Vit C | 66.2 ± 3.9+ | 70.7 ± 3.3+ | 5.7 ± 0.4+
G5 + Vit E | 69.5 ± 3.9+ | 73.5 ± 3.3+ | 6.0 ± 0.4+
G6 + A+C+E | 80.3 ± 3.7+‡ | 84.7 ± 3.2+‡ | 7.1 ± 0.4+‡

*p < 0.001 vs. G1; +p < 0.05 vs. G2; ‡p < 0.05 vs. G3-G5.

DISCUSSION

The pronounced elevation of ovarian MDA and concurrent loss of GSH, SOD, and CAT in ZnO-NP-treated rats reflect excessive ROS generation via Zn2+ leaching and mitochondrial electron transport chain disruption [6,7]. The resultant oxidative stress upregulates pro-apoptotic Bax via p53 and downregulates anti-apoptotic Bcl-2, promoting mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, caspase-3 activation, and internucleosomal DNA fragmentation [13-17,23-25].

Microvascular endothelial damage manifested as vascular congestion and stromal edema, while damage-associated molecular patterns (DAMPs) triggered innate immune infiltration and NLRP3 inflammasome activation [26,27]. Granulosa cell apoptosis caused follicular atresia and premature luteolysis, precipitating a hypergonadotropic-hypogonadal endocrine profile (decreased estradiol and progesterone, loss of negative feedback triggering FSH/LH surges) [8-10,28-31]. Downstream embryonic impairment (reduced implantation, cleavage retardation) reflects combined oocyte oxidative damage and defective endometrial decidualization [21,32-36].

The synergistic superiority of the combined A+C+E regimen arises from distinct yet cooperative chemical and biological mechanisms: lipophilic vitamin E interrupts membrane lipid-peroxidation; hydrophilic vitamin C scavenges aqueous ROS and regenerates oxidized alpha-tocopherol; and vitamin A stabilizes cell membranes and modulates gene expression via nuclear retinoic acid receptors (RARs) [18-22] (Figure 4).

Figure 4

CONCLUSION

ZnO-NPs cause multi-level female reproductive toxicity via oxidative stress-induced activation of the intrinsic Bax/Bcl-2/caspase-3 apoptotic pathway in granulosa cells, leading to follicular atresia, endocrine disruption, and early embryonic impairment. Combined supplementation with vitamins A, C, and E provides comprehensive, synergistic histoprotective and antioxidant effects, near-completely restoring ovarian cytoarchitecture, hormonal homeostasis, and early reproductive outcomes.

Abbreviations & Declarations

ABBREVIATIONS

CAT: Catalase

DAMPs: Damage-Associated Molecular Patterns

DLS: Dynamic Light Scattering

E2: 17beta-Estradiol

FSH: Follicle-Stimulating Hormone

GD: Gestational Day

GSH: Reduced Glutathione

HPG: Hypothalamic-Pituitary-Gonadal

IHC: Immunohistochemistry

LH: Luteinizing Hormone

MDA: Malondialdehyde

MOMP: Mitochondrial Outer Membrane Permeabilization

P4: Progesterone

ROS: Reactive Oxygen Species

SOD: Superoxide Dismutase

TBARS: Thiobarbituric Acid Reactive Substances

TEM: Transmission Electron Microscopy

TUNEL: Terminal deoxynucleotidyl transferase dUTP Nick End Labeling

XRD: X-Ray Diffraction

ZnO-NPs: Zinc Oxide Nanoparticles

ACKNOWLEDGMENTS

The authors gratefully acknowledge the technical assistance of the staff of the Animal House Facility, the Central Research Laboratory, and the Department of Histopathology at the Kut Technical Institute, Middle Technical University, particularly for expert support with IHC and TUNEL procedures.

DECLARATIONS

Ethical Approval: Approved by the Institutional Animal Ethics Committee of the University of Wasit, College of Science, Iraq (Ethical Approval No. WU-2125).

Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflicts of Interest: The authors declare no conflict of interest.

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