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.

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).

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).

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.