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Article

Concentration-Related Ultrastructural Alterations in Mouse Oocytes Following In Vitro Lindane Exposure

by
Marta Gatti
1,†,
Manuel Belli
2,3,†,
Mariacarla De Rubeis
4,
Stefania Annarita Nottola
4,
Guido Macchiarelli
5,
Carla Tatone
5,
Giovanna Di Emidio
5 and
Maria Grazia Palmerini
5,*
1
Research Unit of Immunorheumatology, Department of Medicine and Surgery, School of Medicine, University of Rome “Campus Biomedico”, 00128 Rome, Italy
2
Department of Human Sciences and Promotion of the Quality of Life, San Raffaele Roma Open University, 00166 Rome, Italy
3
Laboratory of Molecular, Cellular and Ultrastructural Pathology, IRCCS San Raffaele Roma, 00166 Rome, Italy
4
Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Sapienza University, 00161 Rome, Italy
5
Department of Life, Health and Environmental Sciences, University of L’Aquila, 67100 L’Aquila, Italy
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Appl. Sci. 2025, 15(15), 8320; https://doi.org/10.3390/app15158320
Submission received: 13 June 2025 / Revised: 16 July 2025 / Accepted: 23 July 2025 / Published: 26 July 2025
(This article belongs to the Special Issue Exposure Pathways and Health Implications of Environmental Chemicals)
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Abstract

Lindane, a persistent organochlorine pesticide, exerts toxic effects on the female reproductive system, compromising oocyte quality and maturation. However, the effects of this pesticide on mammalian oocyte morphology and ultrastructure remain unknown. This study investigated the effects of Lindane on mouse oocyte ultrastructure using an in vitro model with Transmission Electron Microscopy (TEM) at concentrations from 1 to 100 μM. The results revealed a progressive dose-related trend of alterations: at 1 μM, mild swelling of smooth endoplasmic reticulum (SER) vesicles; at 10 μM, increased SER dilation and cytoplasmic disorganization; and at 100 μM, pronounced vacuolization, mitochondrial swelling, dense lamellar bodies (dlbs), and multivesicular bodies (MVBs) indicative of autophagic activity. Mitochondrial alterations increased significantly with concentration: 3.2 ± 0.8 (control), 5.7 ± 1.0 (1 μM), 9.4 ± 1.5 (10 μM), and 16.8 ± 2.3 (100 μM) altered mitochondria per oocyte (p < 0.01). Vacuole frequency was notably elevated at 100 μM (4.3 ± 1.1 vs. 0.7 ± 0.5 in controls), and mislocalization of organelles within the ooplasm was observed. In conclusion, Lindane-induced oocyte ultrastructural alterations were observed at all tested concentrations but were more pronounced at 100 μM. These results highlight its impact on female fertility and may guide the search for protective agents, as well as efforts to reduce environmental exposure to endocrine disruptors.

1. Introduction

Pesticides, which can be naturally derived, industrially manufactured, or produced from microorganisms, are widely used in agriculture to control weeds and insect pests [1,2]. However, their extensive use has led to environmental contamination, raising public health concerns. Many pesticides persist in the food chain and environmental sources like water, air, soil, and sediments, resulting in continuous exposure with potential short- and long-term risks to humans and wildlife [3,4,5].
In recent decades, female infertility has significantly increased, with evidence suggesting a strong link between environmental pollutants and reproductive health issues, in addition to genetic and lifestyle factors [6,7,8,9]. As a result, increasing attention is being given to the effects of pollutants on female fertility, pregnancy outcomes, and overall reproductive health [10,11].
Organochlorine pesticides (OCPs) are persistent, lipophilic chemicals widely used in agriculture and epidemics [12,13,14,15]. They accumulate in fatty tissues like the ovaries and liver and pose risks including carcinogenicity, neurotoxicity, genotoxicity, and disruption of endocrine, reproductive, and immune systems, even at low environmental exposures [16,17,18,19].
Lindane, the isomer γ-Hexachlorocyclohexane, is one of the longest-lasting OCPs, used as a broad-spectrum insecticide and also for medical and veterinary purposes, employed for the treatment of scabies and parasites [4,12]. Recent evidence of its toxicity has led to the classification of Lindane as carcinogenic to humans (Group 1) by the International Agency for Research on Cancer (IARC) based on statistically significant increases in the risk of non-Hodgkin lymphoma with higher levels of occupational exposure [20]. In the general population, blood concentrations of Lindane have been reported in the range of 0.1–1 µg/L [21,22,23]. Neurologic symptoms and changes in liver function have been observed among workers when blood concentrations exceed 20 µg/L [2,24,25,26].
Due to its high toxicity and persistence, Lindane has been banned or restricted in many countries, with a global agricultural ban enforced by the 2009 Stockholm Convention. Italy banned its use only in 2022. Despite these measures, some countries like China, India, the US, and Canada still use Lindane for treating lice and scabies, with India being the sole producer for pharmaceutical purposes. Lindane production has created vast toxic waste, causing environmental contamination [27,28,29]. Currently, the only active production facility is in India, uniquely for pharmaceutical purposes [30]. Although its use has declined, residues persist, and global trade in treated products continues to pose exposure risks [3,31,32]. Furthermore, research on occupational exposure is limited, and there is a lack of direct data in those areas where it is still in use [2].
Lindane is classified as an endocrine disruptor and reproductive toxicant, interfering with hormone function, synthesis, and receptor activity. Due to its lipophilic nature, it can accumulate in breast milk and placenta and has been associated with female infertility, intrauterine growth retardation, and preterm births [3,4,16]. Its reproductive toxicity is linked to gap junction disruption, altered calcium homeostasis, impaired maturation-promoting factor (MPF) activity, and spindle formation. Specifically, it has been shown to inhibit connexin-43-mediated gap junction formation in granulosa cells, suppressing FSH- and TGF-β1-induced steroidogenesis and intercellular connectivity [33]. Moreover, Lindane exposure abolishes oocyte-directed follicle-organizing activity by disrupting gap junctions between the oocyte and surrounding somatic cells, thus impairing essential cumulus–oocyte communication [34]. These effects are further supported by in vivo and in vitro studies demonstrating that Lindane exposure interferes with oocyte maturation and preimplantation development [4,33,34,35], likely through the inhibition of molecular signaling pathways that rely on intact gap junctions [4,22,33,36].
In vivo studies in mice showed mild reproductive effects in offspring, such as increased uterine weight, earlier vaginal opening, and smaller primary oocytes, possibly involving the ERβ pathway [3].
In vitro, Lindane impaired embryo development beyond the eight-cell stage in mice and reduced morula/blastocyst formation in bovine oocytes, despite normal fertilization and cleavage rates [37,38]. Previous results also showed ultrastructural damage in mouse granulosa cells related to increasing Lindane concentrations, including nuclear membrane invagination, cytoplasmic blebbing, and loss of intercellular connections [39].
Currently, no ultrastructural data exist on Lindane’s effects on mammalian oocytes, limiting understandings of its impact on fertility. Given Lindane’s persistence in the environment, ongoing human exposure risk, and known endocrine-disrupting properties, this study aimed to fill this knowledge gap by examining morphological and ultrastructural changes in mouse oocytes. TEM, a powerful tool for detailed observation at organellar and sub-organellar levels, enabled the identification of fine physiological and pathological alterations, often undetectable with conventional imaging modalities. Using an in vitro model, we explored the effects on ultrastructure of mouse oocytes exposed to increasing concentrations of Lindane through Light Microscopy (LM) and TEM. This approach allowed us to evaluate the pesticide’s effects on oocyte ultrastructure, identify specific cellular compartment alterations, and investigate the relationship between Lindane-induced ultrastructural damage and reproductive toxicity, clarifying its role as a reproductive toxicant [40,41,42].

2. Materials and Methods

2.1. Chemicals

Unless otherwise stated, all materials were purchased from Sigma Chemical (St. Louis, MO, USA).

2.2. Animals

Swiss CD1 female mice (Harlan, Udine, Italy) were housed individually under controlled environmental conditions (12 h light/dark cycle, 21 ± 1 °C), with ad libitum access to food and water. Twelve prepubertal females (aged 21–23 days) received an intraperitoneal injection of 5 IU Pregnant Mare Serum Gonadotropin (PMSG; Intervet, Milan, Italy) to induce follicular development, and were euthanized 48 h later by cervical dislocation. All procedures were conducted in accordance with the guidelines of the Italian Ministry of Health for the care and use of laboratory animals and were approved by both the University of L’Aquila Institutional Animal Care Committee and the Italian Ministry of Health (Authorization No. 329/2022-PR).

2.3. In Vitro Maturation (IVM), Oocyte Isolation, and Experimental Protocol

Cumulus–oocyte complexes (COCs, 15/group) were collected by puncturing antral follicles from mouse ovaries using an insulin syringe. They were then subjected to in vitro maturation (IVM) at 37 °C in 5% CO2 in air, using alpha MEM medium supplemented with 0.23 mM pyruvate and 2 mM L-glutamine [43]. Lindane (PESTANAL, 45548) was dissolved in dimethyl sulfoxide (DMSO), and serial dilutions were prepared in culture medium to achieve the desired concentrations. In our study, DMSO was used at concentrations ≤0.1% as a vehicle control in accordance with previous studies on Lindane and other organochlorines [44]. These studies have reported that DMSO at such concentrations does not induce morphological, maturation, or ultrastructural changes in oocytes or granulosa cells [45,46].
To investigate Lindane toxicity, the concentrations used (1 μM, 10 μM, and 100 μM) were based on previous reproductive toxicity studies [38,39,47]. Despite exceeding typical human and occupational exposure [21,22,23,24,25,26], these concentrations reflect Lindane bioaccumulation in lipid-rich tissues such as the reproductive system and mimic chronic exposure.
COCs were casually assigned to four experimental groups—Control, Lindane 1 μM (L1), Lindane 10 μM (L10), Lindane 100 μM (L100)—and subjected to IVM [39]. After 16 h, metaphase II (MII) oocytes were isolated by removing cumulus cells. All experiments were performed in triplicate.

2.4. Preparations for LM and TEM

Denuded oocytes were fixed in 2.5% glutaraldehyde (Agar Scientific, Cambridge Road Stansted Essex, Cambridge, UK) in PBS (pH 7–7.4) at 4 °C for at least 48 h, following established protocols [48,49]. They were then post-fixed in 1% osmium tetroxide (Electron Microscopy Sciences, 1560 Industry Road, Hatfield, PA, USA), embedded in 1% agar and EMbed-812 epoxy resin (Electron Microscopy Sciences, 1560 Industry Road, Hatfield, PA, USA), and sectioned using an Ultracut E ultramicrotome (Leica EMUC6, Wetzlar, Germany).
Semithin sections (1 μm thick) were stained and examined by LM, while ultrathin sections (70–90 nm thick) were stained with Uranyless (TAAB Laboratories Equipment Ltd., Aldermaston, UK) and Lead Citrate (Electron Microscopy Science, 1560 Industry Road, Hatfield, PA, USA) for TEM imaging using Philips CM100 (Eindhoven, Holland) and JEOL 1400 PLUS (Tokyo, Japan) electron microscopes operating at 100 kV. Based on previous studies [50,51], TEM was used to qualitatively assess oocyte morphology and ultrastructure, including organelle type and distribution, ooplasmic vacuolization, cortical granule (CG) density and positioning, microvilli structure, oolemma integrity, and zona pellucida (ZP) appearance.

2.5. Morphometric Analysis

Low-magnification TEM images of the control and Lindane-treated oocytes underwent morphometric analysis using ImageJ version 1.54 (https://imagej.net/ij/, accessed 30 January 2020). The numerical density of mitochondria, large SER vesicles (≥0.5 µm diameter), MVBs, and dense lamellar bodies was measured.
For each group, at least five equatorial sections per oocyte (with 3 μm intervals) from three oocytes were analyzed. The results are reported as numerical density per 50 μm2 of oocyte area [45].
CG and microvillar densities were assessed on TEM micrographs at 2500× magnification, examining the entire surface profile on five equatorial sections from three oocytes per group. Data are expressed as the number of CGs and microvilli per 10 μm of oocyte linear surface profile.

2.6. Statistical Analysis

All data are expressed as means ± standard deviation (SD). Statistical comparisons were performed using one-way ANOVA with Tukey’s honest significant difference (HSD) tests for post hoc analysis (GraphPad InStat. GraphPad Software, version 10.4.2, La Jolla, San Diego, CA, USA). Before performing one-way ANOVA followed by Tukey’s post hoc test, data distribution was assessed for normality using the Shapiro–Wilk test and for homogeneity of variance using Levene’s test. All datasets met the assumptions required for parametric analysis. Differences in values were considered significant if p < 0.05.

3. Results

For each experimental group, the microtopographical and ultrastructural description of cellular organelles, based on electron microscopy analysis, will be presented in the following order, starting from the inner cytoplasmic region of the oocyte and moving outward toward the cortex and external layers: mitochondria, SER, MVBs and multivesicular aggregates (MVAs), CGs, microvilli, and ZP.

3.1. Controls

LM showed the control group to have a regular round shape. The ooplasm appeared dense, with a homogeneous distribution of organelles, surrounded by an intact ZP (Figure 1A, inset).
TEM micrographs showed abundant organelles uniformly distributed throughout the ooplasm (Figure 1A). The most numerous and commonly identified organelles were mitochondria.
Mitochondria presented a round or oval shape, characterized by evident cristae, dense mitochondrial matrix, and distinguishable inner and outer mitochondrial membranes. Sometimes, a large clear vesicle occupied a part of the matrix (Figure 1A,B). Cytoplasmic lattice, a typical fibrillar structure of mouse oocyte ooplasm, appeared striated and scattered in the ooplasm (Figure 1C).
Tubular elements of SER were seen in the ooplasm (Figure 1C,D). MVA and MVBs of different sizes were mostly seen close to the plasma membrane (Figure 1B,D). Occasionally, dense lamellar bodies, with a configuration of concentric circles, were found in the ooplasm, together with small and distinct vesicles (Figure 1D).
Numerous round and dark electron-dense CGs linearly occupied the peripherical region of the oocyte, just beneath an uninterrupted oolemma (Figure 1D).
Long and thin microvilli projected from the oolemmal surface to the perivitelline space (PVS). Furthermore, ultrastructural analysis evidenced a continuous and dense ZP and the PVS was uniform (Figure 1D).
Table 1 reports a summary of the qualitative analysis.

3.2. Lindane 1 µM (L1)

By LM, oocytes exposed to Lindane 1 µM had a rounded shape, an intact ZP, and a narrow PVS (Figure 2A, inset).
By TEM, at low magnification, the organelle density and distribution seemed slightly reduced, compared to controls. The fibrillar structures of the cytoplasmic lattice were commonly dispersed throughout the ooplasm (Figure 2A).
Mitochondria, usually spherical or ovoid, were the most numerous organelles in this experimental group; their cristae were recognizable on the periphery and parallel to the outer mitochondrial membrane. They appeared mostly organized in clusters, with a double-layered electron-dense mitochondrial membrane and a homogeneous matrix, as in the control group (Figure 2B). Frequently, mitochondria presented a vacuole or prominent electron-dense granules in their matrix (Figure 2B,D,E); however, the density of vacuolized mitochondria was almost unchanged compared to the control group (Figure 2B,D,E) (Table 1).
MVA and MVBs were found located in the cortical region of the ooplasm, near the plasma membrane (Figure 2C,D); dense lamellar bodies were occasionally detected in the ooplasm (Figure 2C inset). Tubular and vesicular SER elements, sometimes dilated and adjacent to mitochondria, were widespread in the ooplasm (Figure 2C). However, some areas appeared with a lower organelle density with respect to the controls.
Below the oolemma, the CGs had an electron-dense and rounded appearance. Overall, a reduced amount of irregularly distributed CGs were present in the L1 group compared to controls (Figure 2B–D) (Table 1).
The PVS appeared narrow, with a different distribution of microvilli with respect to what was observed in the control group. Among areas of long and thin microvilli, clusters of thicker and shorter microvilli were often found (Figure 2B,C inset). Extracellular material, exocytic vesicles, and cell fragments were detected in the PVS (Figure 2C). The ZP was thin, dense, and continuous (Figure 2A–C) (Table 1).

3.3. Lindane 10 µM (L10)

After exposure to Lindane 10 µM, mouse oocytes presented a round-to-ovoid shape by LM, with a narrow PVS and an intact ZP (Figure 3A, inset).
The ultrastructural examination revealed an ooplasm with a lighter texture compared to previous groups. In general, organelle density diminished with respect to the control group, being more similar to what was observed in the L1 group (Figure 3A).
Ultrastructural changes were seen in some subcellular structures. Fibrillar structures, termed cytoplasmic lattices, densely filling the oocyte cytoplasm were frequently observed (Figure 3B).
Round mitochondria appeared reduced, with fewer electron-dense mitochondrial cristae, compared with control and L1 groups (Figure 3A,C,E). Sometimes, they presented vacuoles of different sizes or electron-dense granules in their matrix (Figure 3E). They were unevenly distributed in the ooplasm, as isolated elements or clusters (Figure 3A,B). Occasionally, swollen mitochondria were observed (Figure 3A) (Table 1).
MVAs were mostly distributed in the ooplasm cortex, frequently accompanied by numerous dense lamellar bodies and lipid droplets (Figure 3B–D). Several large and dilated SER vesicles were sparsely diffused in the ooplasm, with the occasional presence of electron-dense material in the lumen, related to the ER stress (Figure 3B,D,F).
Rounded CGs, with an electron-pale content, formed a discontinuous rim in the cortex. Their numerical density appeared reduced if compared to the control and L1 groups (Figure 3A) (Table 1).
Similarly to what was observed in the L1 group, the oolemma showed areas deprived of microvilli. The microvilli, shorter, thicker, and smaller, were irregularly distributed and less numerous (Figure 3A–C) with respect to the controls. Extracellular material, cell fragments, and debris were detected in the PVS (Figure 3C). The ZP appeared continuous and dense (Table 1).

3.4. Lindane 100 µM (L100)

Oocytes exposed to L100 showed significant ultrastructural alterations. By LM, the oocytes were seen to have a round shape, with an intact ZP and the presence of numerous vacuoles (Figure 4A, inset). TEM analysis revealed overall alterations, after comparison to the previous groups and especially to the controls, in the type of organelles present, their distribution, and their quantity (Figure 4A).
The numerosity of mitochondria appeared drastically reduced; they were usually roundish or ovoid, with an inhomogeneous pattern distribution in the ooplasm, occasionally associated with vacuoles. Matrix density also appeared less intense (Figure 4A,B). Cytoplasmic lattices were identifiable by fibrillar structures; numerous vesicles attributable to SER dilation were present in the ooplasm, with electron-dense material in the lumen, suggesting ER stress leading to pronounced vacuolization (Figure 4B,C).
Evident vacuolization was observed in this group (Figure 4A,B). Spherical vacuoles appeared generally empty, surrounded by a single membrane (Figure 4E). Swollen MVBs were mostly located in the cortex, near the plasma membrane, accompanied sometimes by autophagic-like vesicles (Figure 4C, inset). Dense lamellar bodies, sometimes containing flocculant material, were numerous. They were occasionally close to MVBs, forming organelle clusters. Notably, two dense lamellar bodies enclosed in a marked lumen bordered by a single membrane were frequently observed (Figure 4B, inset).
Furthermore, TEM analysis showed structures compatible with mature autophagic vesicles, enclosed by a single membrane and containing membranous material of unrecognizable origin, dispersed in the ooplasm (Figure 4B,C, inset; Figure 4D). Some regions just beneath the oolemma showed organelle-free zones.
CGs were almost absent in the cortical region of the oocytes. Although the oolemma was continuous, the microvillar coverage diminished, also with short and thick microvilli (Figure 4C,D).
In the PVS, extracellular materials, such as debris, extracellular vesicles, and cell fragments, were found. Notably, dense lamellar bodies were observed in the PVS, likely originating from degenerating, altered microvilli (Figure 4D). The ZP appeared dense.
Table 1 reports a summary of the morphological qualitative analysis.

3.5. Morphometric Analysis

The morphometric analysis evidenced a downward trend in the mitochondrial numerical density from control to increasing concentrations of Lindane. In detail, the mean number of mitochondria per 50 µm2 was 20.6 ± 5.1, 19.8 ± 3.7, 17 ± 5.6, and 8.2 ± 2.4 in the control, L1, L10, and L100 groups, respectively. The numerical density of mitochondria significantly dropped in the L100 group when compared with controls and the L1 group (8.2 ± 2.4 vs. 20.6 ± 5.1; 19.8 ± 3.7; p < 0.001), and a significant reduction was detected between the L100 and L10 groups (8.2 ± 2.4 vs. 17 ± 5.6; p < 0.05). No significant differences were present among the L1 and L10 groups or when comparing the control vs. L1 and L10 groups (Table 2).
The amount of large SER vesicles did not show significant differences between all experimental groups, even if a rising trend was observed from the control to L100 group. In detail, the mean number of large SER vesicles per 50 µm2 was 5.6 ± 1.7, 6.8 ± 3.2, 7 ± 1.6, 9.8 ± 2.4 (p > 0.05) in the control, L1, L10 and L100 groups, respectively.
In contrast, the numerical density of MVBs and dense lamellar bodies significantly increases in the L100 group with respect to all other experimental groups (p < 0.0001). Significant differences were also found in the control group when compared to the L10 group (p < 0.001). In detail, the mean number of MVBs and dense lamellar bodies per 50 μm2 was 3.6 ± 0.5 (controls), 4.4 ± 0.9 (L1), 6 ± 1 (L10), and 9.2 ± 1.3 (L100), also showing an increasing trend.
The morphometric evaluation of CGs showed a progressive reduction in their numerosity after exposure to increasing concentrations of Lindane, even if it was not significant. In detail, the mean number of CGs per 10 µm of linear surface was 2.6 ± 1.1, 2 ± 1.6, 1.6 ± 1.5, and 1 ± 0.7 in the control, L1, L10, and L100 groups, respectively (p > 0.05).
The number of microvilli per 10 µm of linear surface decreased at increasing concentrations of the pesticide, with a significant difference between the groups exposed to 10 μM and 100 μM concentrations (8.2 ± 1.5; 8.6 ± 1.1; p < 0.05) when compared to controls (13 ± 2.7) (Table 2).

4. Discussion

Lindane, a pesticide commonly used on forage crops, is a known reproductive toxicant. It alters the endocrine system, altering estrogen–progesterone balance and affecting reproductive health [52,53]. It has shown reproductive toxicity in both males and females across species, even at low doses (0.25 mg/kg/day, or 1/1000th of its LD50) [4,54,55,56]. Its lipophilicity allows it to bioaccumulate in biological fluids like follicular fluid, maternal and cord blood [22,57,58], and breast milk, increasing women’s exposure risk [59,60,61]. Even with brief preincubation, Lindane impaired the growth of isolated primordial germ cells in culture, potentially due to apoptotic cell death, as indicated by increased caspase-3 activity and reduced AKT kinase phosphorylation [44]. An additional in vitro study showed that Lindane concentrations between 10 and 100 μM interrupted the formation of the first meiotic spindle and polar body extrusion in mice oocytes [62].
Our work examined the impact of increasing concentrations of Lindane on the ultrastructure of mouse oocytes in an in vitro model. To the best of our knowledge, no prior studies have explored the effects of this compound on the ultrastructure of mammalian oocytes. At a concentration of 1 μM, Lindane caused minor morphological changes in mouse oocyte ultrastructure, including a slight decrease in mitochondria and CGs within the ooplasm, along with irregular microvilli. These early adverse effects became more evident at 10 μM, with notable reductions in organelle numerical density (particularly mitochondria), SER dilation, altered CG distribution, and changes in microvilli structure and quantity. At the highest concentration tested (100 μM), pronounced ooplasmic alterations were observed, including a significant reduction in mitochondria and CGs, pronounced microvilli thinning, cytoplasmic vacuolization, and SER dilation. The presence of autophagic vesicles and numerous secondary lysosomes indicates a potential cellular stress response. These findings demonstrate dose-related variations in ooplasmic organelle composition and morphology, suggesting impaired oocyte maturation or viability, consistent with previous studies [50,63,64].

4.1. Mitochondrial Alterations and Cytoskeleton Impairments

Several studies have demonstrated that Lindane and other organochlorine pesticides can impair mitochondrial membrane potential, reduce ATP production, and promote mitochondrial swelling and degeneration through oxidative stress mechanisms and bioaccumulation in ovarian tissues [65,66,67,68,69]. For instance, Zhurabekova et al. (2018) and Malott and Luderer (2021) reported a significant decrease in mitochondrial activity and structural integrity in oocytes and ovarian cells exposed to these toxicants, linked to disrupted oxidative phosphorylation and energy failure [23,70]. Changes in mitochondrial shape, number, distribution, and function are linked to cell metabolism and differentiation. Mitochondrial movement and multiplication depend on the cytoskeleton, which is crucial during oocyte growth. In high-quality oocytes, mitochondria are numerous and evenly distributed, while in lower-quality oocytes, they tend to group [71,72]. Loss or alteration of mitochondrial arrangement indicates cytoskeletal disruption and cytoplasmic immaturity, leading to oocyte maturation failure and fertilization issues [73]. These molecular data support the hypothesis that the observed ultrastructural mitochondrial alterations represent a downstream manifestation of a complex toxicant-induced mitochondrial dysfunction, ultimately contributing to impaired oocyte quality and female infertility. Notably, mitochondria exposed to Lindane exhibited electron-dense granules, suggesting pesticide-induced stress and dysfunction. Such granule accumulation has been previously linked to numerous pathological conditions and mitochondrial impairments [74,75].

4.2. ER Dilatation, Vacuole Formation, and Autophagy as Markers of Oocyte Stress

Our ultrastructural data revealed a significant change in the morphology of SER vesicles following exposure to Lindane, with notable dilation observed at both 1 μM and 10 μM concentrations. However, at the highest concentration (100 μM), this dilation was more pronounced and accompanied by vacuoles in the ooplasm. Given the critical role of the ER in calcium homeostasis, protein synthesis, and fertilization-related signaling, its structural disruption is indicative of ER stress [76,77], affecting oocyte maturation in response to toxicants, as previous studies reported [78].
In line with these findings, toxicological studies have increasingly demonstrated that exposure to OCPs such as Lindane may lead to alterations in ovarian function through complex molecular pathways. These compounds have been shown to induce oxidative stress, mitochondrial dysfunction, and ER stress, which synergistically activate intrinsic apoptotic cascades in oocytes and surrounding granulosa cells [65]. Lindane has been shown to upregulate pro-apoptotic proteins and cause ultrastructural alterations in both mitochondria and the ER, interfering with oocyte maturation and triggering follicular atresia as a result [58]. These mechanistic insights reinforce the hypothesis that environmental toxicants serve as pro-apoptotic agents within the ovarian microenvironment, contributing to infertility and early ovarian failure in exposed populations.
In MII oocytes, vacuoles are rare and may indicate cytoplasmic immaturity or aging [74,79]. Vacuolization is a common response to injury in mature oocytes, resulting from the swelling and merging of SER vesicles [80,81]. Since Lindane affects calcium homeostasis, high concentrations likely induce ER stress, leading to an imbalance in intracellular calcium, causing dilated SER vesicles to turn into prominent vacuoles [82].
Following exposure to the highest Lindane concentration (100 μM), there was a significant increase in dense lamellar bodies and MVBs, identified as secondary lysosome-like structures, in the ooplasm of mouse oocytes [83,84]. These structures function as autophagic vacuoles or autolysosomes, facilitating the breakdown of internal components and indicating enhanced autophagic activity due to cellular stress. Furthermore, the presence of extracellular debris in the perivitelline space further supports the occurrence of autophagic processes, highlighting degenerative changes linked to autophagic exocytosis and apoptosis [85,86].

4.3. Effects on CG and Microvilli Distribution

CGs, essential for preventing polyspermy during fertilization, are important markers of oocyte quality and cytoplasmic maturation and are used to assess oocyte maturity and organelle organization [87]. Our data, although not statistically significant, suggest that Lindane exposure prompted the dose-related reduction and misdistribution of CGs. This could impair the oocyte’s ability to prevent polyspermy, leading to potential fertilization issues and infertility [88,89,90]. These results align with previous research showing that organochlorines can cause polyspermy by altering the zona pellucida. Additionally, the observed changes in CG distribution indicate that Lindane may alter the cytoskeleton, which regulates CG movement [87].
Moreover, results from this work showed irregular and reduced microvilli distribution after Lindane exposure, which plays a key role in sperm–oocyte interactions [91,92,93], during fertilization. Alteration in the peripheral oolemma may result in diminished oocyte quality and an increased risk of fertilization failure.

4.4. Hypothesis on Role of Cumulus Cells in Lindane-Induced Oocyte Damage, Study Limitations, and Future Perspectives

In our recent in vitro study on mouse oocytes exposed to the endocrine disruptor Mancozeb, we observed significant ultrastructural changes only at the highest concentration (1 μg/mL), suggesting that cumulus cells may have a protective role toward the oocytes [43]. In contrast, this current work revealed ultrastructural alterations in mouse oocytes exposed to Lindane across all experimental groups, with more severe damage observed in the L100 group.
It is well-established that cumulus cells surround oocytes, forming COCs, and are essential in oocyte maturation and fertilization through bidirectional communication and metabolic interdependence [94,95]. Acting as a biological barrier, cumulus cells rely on gap junctions formed by connexin proteins, such as Cx43, to facilitate the exchange of small molecules. This connection supports the transfer of metabolites and regulatory molecules, enhancing cytoplasmic maturation and aiding early embryo development through coordinated cellular changes [96,97].
Considering the Lindane-induced inhibition of gap junctional intercellular communication, as previously demonstrated in numerous studies [34,98], the changes here observed in mouse oocytes may be associated with its activity as a gap junction blocker, thus explaining its possible entry into the oocyte to exert its cytotoxic effect. Results from this study evidenced the action of Lindane in affecting the bidirectional communication between cumulus cells and oocytes by abolishing gap junctions and interrupting the functional and protective role of cumulus cells toward the oocyte. However, due to the limited research on Lindane, further studies are needed to fully explore its reproductive toxicity and comprehensively examine the specific mechanisms underlying its effects on reproductive health and related pathologies. The limited availability of comparable ultrastructural data on Lindane exposure, particularly in mammalian oocytes, also restricts broader comparative analysis.
Recent applications of artificial intelligence and recurrent neural networks in monitoring and diagnostic fields, such as the study by Pratticò et al. (2025), which integrates LSTM and U-Net models for anomaly detection in electrical systems, underscore the potential of deep learning techniques for identifying complex patterns. In the future, a similar approach could be applied to the automated analysis of ultrastructural images, aiming to improve the early detection of toxic effects induced by environmental pollutants on oocytes [99].

5. Conclusions

In conclusion, this study demonstrates an increasing dose-related trend in the reproductive toxicity of Lindane on mouse oocyte ultrastructure, with the most severe alterations, such as vacuolization, mitochondrial damage, and SER dilation, occurring at the highest concentration (100 μM). This ultrastructural evidence, presented here for the first time, underscores Lindane’s harmful effects on oocyte morphology and organelle integrity.
These alterations may serve as early indicators of compromised oocyte quality and provide a morphological basis for identifying sublethal toxicological effects.
Our findings are critical for understanding the impact of environmental toxicants on female fertility, offering a morphological framework for future studies regarding reproductive risk assessments and supporting the use of TEM as a complementary tool for evaluating oocyte integrity.
Furthermore, the reproducible in vitro model provides a useful system to explore toxicant-induced damage and evaluate potential protective compounds, including antioxidants and therapeutic agents. This approach also represents an important future direction for toxicological screening correlated with infertility.

Author Contributions

Conceptualization, M.G., S.A.N., G.M. and M.G.P.; methodology, M.G.P., M.G., M.B. and M.D.R.; validation, M.G. and M.G.P.; formal analysis, M.G., M.B. and M.G.P.; investigation, M.G. and M.G.P.; resources, S.A.N. and G.M.; data curation, M.G. and M.G.P.; writing—original draft preparation, M.G. and M.G.P.; writing—review and editing, M.G., M.B., S.A.N., G.M. and M.G.P.; visualization, G.D.E., C.T., S.A.N. and G.M.; supervision, G.D.E., C.T., S.A.N. and G.M.; project administration, M.G.P., S.A.N. and G.M.; funding acquisition, M.G.P., S.A.N. and G.M. All authors have read and agreed to the published version of the manuscript.

Funding

The present study was supported by grants from the University of L’Aquila, L’Aquila (fondi RIA 2012–2015), and Sapienza University, Rome (Bandi SEED-PNR 2021–2022).

Institutional Review Board Statement

This study was approved by the Internal Review Board of the University of L’Aquila (authorization n. 329/2022-PR, released on 30 May 2022).

Data Availability Statement

The data presented in this study are available on request from the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The ultrastructure of mouse oocytes in the control group. Representative TEM micrographs showing (A) the general morphology in MII-stage mouse oocytes of intracellular organelles and microvillar processes. Round/ovoid mitochondria (m) and cortical granules (CGs) are visible; ZP: zona pellucida (TEM, bar: 2 µm). Inset in A: A representative image of a semithin section of a mouse oocyte (LM, mag: 40×). (B) The cortex of a mouse oocyte with multivesicular aggregates (MVAs), ovoid mitochondria (m), and a regular distribution of microvilli (mv) on the oolemma (TEM, bar: 2 μm). (C) A portion of ooplasm showing cell organelles at high magnification: mitochondria (m) with electron-dense cristae or with the typical vacuole in their matrix (vm), dense lamellar bodies (dlbs), and SER elements. The fibrillar matrix of cytoplasmic lattice (cpl) was abundant in the ooplasm (TEM, bar: 1 µm). (D) An oocyte cortex showing CGs linearly arranged below the oolemma, SER, multivesicular body (MVB), and dense lamellar body (dlb) in close association. mv: microvilli; PVS: perivitelline space; ZP: zona pellucida (TEM, bar: 1 µm).
Figure 1. The ultrastructure of mouse oocytes in the control group. Representative TEM micrographs showing (A) the general morphology in MII-stage mouse oocytes of intracellular organelles and microvillar processes. Round/ovoid mitochondria (m) and cortical granules (CGs) are visible; ZP: zona pellucida (TEM, bar: 2 µm). Inset in A: A representative image of a semithin section of a mouse oocyte (LM, mag: 40×). (B) The cortex of a mouse oocyte with multivesicular aggregates (MVAs), ovoid mitochondria (m), and a regular distribution of microvilli (mv) on the oolemma (TEM, bar: 2 μm). (C) A portion of ooplasm showing cell organelles at high magnification: mitochondria (m) with electron-dense cristae or with the typical vacuole in their matrix (vm), dense lamellar bodies (dlbs), and SER elements. The fibrillar matrix of cytoplasmic lattice (cpl) was abundant in the ooplasm (TEM, bar: 1 µm). (D) An oocyte cortex showing CGs linearly arranged below the oolemma, SER, multivesicular body (MVB), and dense lamellar body (dlb) in close association. mv: microvilli; PVS: perivitelline space; ZP: zona pellucida (TEM, bar: 1 µm).
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Figure 2. The ultrastructure of mouse oocytes in the Lindane 1 µM group. (A) Low-magnification TEM micrographs from MII-stage mouse oocytes. Clustered mitochondria (m), accompanied by SER vesicle and multivesicular aggregates (MVAs) are visible; note the presence of an exocytotic vesicle (arrow) in the perivitelline space (PVS); cpl: cytoplasmic lattice (TEM, bar: 2 µm). Inset in A: A representative semithin section of a mouse oocyte (LM, Mag: 40×). (B) A TEM micrograph of the cortical region in MII mouse oocytes. Clusters of mitochondria (m), with electron-dense cristae and matrix, are visible; MVA: multivesicular aggregates; CGs: cortical granules; mv: microvilli; ZP: zona pellucida (TEM, bar: 1 µm). (C) At higher magnification, generally, the cortex has a reduced organelle density. SER elements of different sizes are dispersed in the ooplasm. Extracellular debris is observed in the PVS (arrowhead); MVAs: multivesicular aggregates; cpl: cytoplasmic lattice; mv: microvilli (TEM, bar: 1 µm). Inset in C: A high-magnification image of a portion of a cortex showing numerous dense lamellar bodies (dlbs) and mitochondria (m) in close association with small and large SER vesicles and the fibrillar matrix of the cytoplasmic lattice (cpl). Note the lack of microvilli (double arrows); ZP: zona pellucida (TEM, bar: 800 nm). (D) Vacuolated round-to-ovoid mitochondria (vm) with electron-dense double membranes, MVAs, and CGs arranged beneath the oolemma (TEM, bar: 800 nm). (E) The details of the mitochondria (m) clusters, also including vacuolated mitochondria (vm), with prominent electron-dense granules (arrows) (TEM, bar: 600 nm).
Figure 2. The ultrastructure of mouse oocytes in the Lindane 1 µM group. (A) Low-magnification TEM micrographs from MII-stage mouse oocytes. Clustered mitochondria (m), accompanied by SER vesicle and multivesicular aggregates (MVAs) are visible; note the presence of an exocytotic vesicle (arrow) in the perivitelline space (PVS); cpl: cytoplasmic lattice (TEM, bar: 2 µm). Inset in A: A representative semithin section of a mouse oocyte (LM, Mag: 40×). (B) A TEM micrograph of the cortical region in MII mouse oocytes. Clusters of mitochondria (m), with electron-dense cristae and matrix, are visible; MVA: multivesicular aggregates; CGs: cortical granules; mv: microvilli; ZP: zona pellucida (TEM, bar: 1 µm). (C) At higher magnification, generally, the cortex has a reduced organelle density. SER elements of different sizes are dispersed in the ooplasm. Extracellular debris is observed in the PVS (arrowhead); MVAs: multivesicular aggregates; cpl: cytoplasmic lattice; mv: microvilli (TEM, bar: 1 µm). Inset in C: A high-magnification image of a portion of a cortex showing numerous dense lamellar bodies (dlbs) and mitochondria (m) in close association with small and large SER vesicles and the fibrillar matrix of the cytoplasmic lattice (cpl). Note the lack of microvilli (double arrows); ZP: zona pellucida (TEM, bar: 800 nm). (D) Vacuolated round-to-ovoid mitochondria (vm) with electron-dense double membranes, MVAs, and CGs arranged beneath the oolemma (TEM, bar: 800 nm). (E) The details of the mitochondria (m) clusters, also including vacuolated mitochondria (vm), with prominent electron-dense granules (arrows) (TEM, bar: 600 nm).
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Figure 3. The ultrastructure of mouse oocytes in the Lindane 10 µM group. (A) A TEM micrograph showing the general morphology of an MII-stage oocyte. Sporadic clusters or isolated mitochondria (m) are irregularly distributed in the ooplasm; mitochondria swelling is observed (arrow). Electron-pale cortical granules (CGs) are detected; dlbs: dense lamellar bodies; vm: vacuolated mitochondria; MVAs: multivesicular aggregates; ZP: zona pellucida. (TEM, bar: 1 µm). Inset in A: An ovoidal-shaped semithin section of a mouse oocyte (LM, Mag: 40×). (B) The cortical region of mouse oocytes showing the patchy distribution of cell organelles. Mitochondria (m) with electron-pale cristae, MVAs, dense lamellar bodies (dlbs), and abundant cytoplasmic lattice (cpl) can be seen. Note the area with a flattened oolemma (double arrows). Numerous dilated SER elements are visible (TEM, bar: 1 µm). (C) The cortex of mouse oocytes shows a reduced numerical density of organelles. Note the presence of extracellular materials and debris (arrowhead) in the PVS. Fewer, shorter, and thicker microvilli (mv) are present alongside dlbs (dense lamellar bodies), MVA, and a large SER vesicle; CGs are absent; (TEM, bar: 1 µm). (D) The details of the vacuolated mitochondria (vm), lipid droplets (lds), and MVAs (TEM, bar: 800 nm). (E) Mitochondria (m) with electron-pale cristae and dense matrix. The arrow indicated prominent granules; cpl: cytoplasmic lattice (TEM, bar: 600 nm). (F) A dilated SER with electron-dense material in the lumen (arrow); MVAs: multivesicular aggregates (TEM, bar: 1 µm).
Figure 3. The ultrastructure of mouse oocytes in the Lindane 10 µM group. (A) A TEM micrograph showing the general morphology of an MII-stage oocyte. Sporadic clusters or isolated mitochondria (m) are irregularly distributed in the ooplasm; mitochondria swelling is observed (arrow). Electron-pale cortical granules (CGs) are detected; dlbs: dense lamellar bodies; vm: vacuolated mitochondria; MVAs: multivesicular aggregates; ZP: zona pellucida. (TEM, bar: 1 µm). Inset in A: An ovoidal-shaped semithin section of a mouse oocyte (LM, Mag: 40×). (B) The cortical region of mouse oocytes showing the patchy distribution of cell organelles. Mitochondria (m) with electron-pale cristae, MVAs, dense lamellar bodies (dlbs), and abundant cytoplasmic lattice (cpl) can be seen. Note the area with a flattened oolemma (double arrows). Numerous dilated SER elements are visible (TEM, bar: 1 µm). (C) The cortex of mouse oocytes shows a reduced numerical density of organelles. Note the presence of extracellular materials and debris (arrowhead) in the PVS. Fewer, shorter, and thicker microvilli (mv) are present alongside dlbs (dense lamellar bodies), MVA, and a large SER vesicle; CGs are absent; (TEM, bar: 1 µm). (D) The details of the vacuolated mitochondria (vm), lipid droplets (lds), and MVAs (TEM, bar: 800 nm). (E) Mitochondria (m) with electron-pale cristae and dense matrix. The arrow indicated prominent granules; cpl: cytoplasmic lattice (TEM, bar: 600 nm). (F) A dilated SER with electron-dense material in the lumen (arrow); MVAs: multivesicular aggregates (TEM, bar: 1 µm).
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Figure 4. The ultrastructure of mouse oocytes in the Lindane 100 µM group. (A) A representative TEM micrograph of MII-stage mouse oocytes showing reduced organelle density. Few mitochondria (m) are visible, grouped mostly in the center of the oocyte; note the presence of moderate vacuolization. Cortical granules (CGs) are absent. v: vacuole; mv: microvilli; ZP: zona pellucida (TEM, bar: 4 µm). Inset in A: A semithin section of mouse oocytes, with few organelles and vacuoles (LM, mag: 40×). (B) A portion of the cortical region with an inhomogeneous organelle distribution. Mitochondria (m) are scarce and isolated, with few evident cristae; CGs are very rare. Dilated SER vesicles are detected; ZP: zona pellucida (TEM, bar: 4 µm). Inset in B: A high-magnification image of an autophagic vesicle, with undistinguishable material, accompanied by double dense lamellar bodies enclosed in a single membrane (arrow) (TEM, bar: 800 nm). (C) A high-magnification image of a cortical area with a higher organelle density; dense lamellar bodies (dlbs), multivesicular bodies (MVBs), large vesicles of SER, and vacuoles (v) are visible. An isolated CG is visible. The microvilli (mv) are thick and irregularly distributed on the oolemma (TEM, bar: 1 µm). Inset in C: Dilated MVAs in close association with dense lamellar bodies (dlbs), and autophagic-like vesicles (avs), at high magnification (TEM, bar: 400 nm). (D) An oocyte cortex showing few cell organelles present. The dense lamellar body (dlb) and MVB are close to autophagic-like vesicles (avs). Dilated SER is present, with unrecognizable electron-dense material in the lumen (arrow). Note the dense lamellar body (dlb) in the PVS (arrowhead), together with extracellular material, exosomes, and debris (*) (TEM, bar: 1 µm). (E) A high-magnification image of a vacuole (v), a dense lamellar body (dlb), and mitochondria (m) with no visible cristae, accompanied by large SER elements; cpl: cytoplasmic lattice (TEM, bar: 800 nm).
Figure 4. The ultrastructure of mouse oocytes in the Lindane 100 µM group. (A) A representative TEM micrograph of MII-stage mouse oocytes showing reduced organelle density. Few mitochondria (m) are visible, grouped mostly in the center of the oocyte; note the presence of moderate vacuolization. Cortical granules (CGs) are absent. v: vacuole; mv: microvilli; ZP: zona pellucida (TEM, bar: 4 µm). Inset in A: A semithin section of mouse oocytes, with few organelles and vacuoles (LM, mag: 40×). (B) A portion of the cortical region with an inhomogeneous organelle distribution. Mitochondria (m) are scarce and isolated, with few evident cristae; CGs are very rare. Dilated SER vesicles are detected; ZP: zona pellucida (TEM, bar: 4 µm). Inset in B: A high-magnification image of an autophagic vesicle, with undistinguishable material, accompanied by double dense lamellar bodies enclosed in a single membrane (arrow) (TEM, bar: 800 nm). (C) A high-magnification image of a cortical area with a higher organelle density; dense lamellar bodies (dlbs), multivesicular bodies (MVBs), large vesicles of SER, and vacuoles (v) are visible. An isolated CG is visible. The microvilli (mv) are thick and irregularly distributed on the oolemma (TEM, bar: 1 µm). Inset in C: Dilated MVAs in close association with dense lamellar bodies (dlbs), and autophagic-like vesicles (avs), at high magnification (TEM, bar: 400 nm). (D) An oocyte cortex showing few cell organelles present. The dense lamellar body (dlb) and MVB are close to autophagic-like vesicles (avs). Dilated SER is present, with unrecognizable electron-dense material in the lumen (arrow). Note the dense lamellar body (dlb) in the PVS (arrowhead), together with extracellular material, exosomes, and debris (*) (TEM, bar: 1 µm). (E) A high-magnification image of a vacuole (v), a dense lamellar body (dlb), and mitochondria (m) with no visible cristae, accompanied by large SER elements; cpl: cytoplasmic lattice (TEM, bar: 800 nm).
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Table 1. Morphological qualitative characterization of MII-stage mouse oocytes after IVM with or without (controls) increasing concentrations of Lindane (1–100 µM).
Table 1. Morphological qualitative characterization of MII-stage mouse oocytes after IVM with or without (controls) increasing concentrations of Lindane (1–100 µM).
ControlLindane 1 μMLindane 10 μMLindane 100 μM
MitochondriaAbundant, round-to-ovoid shape, electron-dense cristaeNumerous, round-to-ovoid shape, electron-pale cristaeLess numerous, round-to-ovoid shape, electron-pale cristaeFew, round-to-ovoid shape, electron-pale cristae, electron-pale matrix
Cortical GranulesNumerous, uniformly distributed, round, electron-denseLess numerous, irregularly distributed, round, electron-denseLess numerous, irregularly distributed, round, electron-denseRare, round, electron-dense
MicrovilliNumerous, long and thinLess numerous, short and thickRare, short and thickRare, short and thick
Zona PellucidaDenseThin and denseThick and denseThick and dense
Table 2. Morphometric comparison of organelle distribution in controls and in Lindane-exposed (1–100 μM) MII-stage mouse oocytes. Values expressed as mean ± SD. Differences in values considered significant if p < 0.05. Morphometry performed using one-way ANOVA with Tukey’s HSD post hoc analysis. Different superscripts indicate statistical significance (p < 0.05).
Table 2. Morphometric comparison of organelle distribution in controls and in Lindane-exposed (1–100 μM) MII-stage mouse oocytes. Values expressed as mean ± SD. Differences in values considered significant if p < 0.05. Morphometry performed using one-way ANOVA with Tukey’s HSD post hoc analysis. Different superscripts indicate statistical significance (p < 0.05).
ControlLindane 1 μM Lindane 10 μMLindane 100 μM
N° of mitochondria/50 μm220.6 ± 5.1 a19.8 ± 3.7 a,c17 ± 5.6 a,c,d8.2 ± 2.4 b
N° of large SER vesicles/50 μm2
(dilated vesicle diameter ≥ 0.5 µm)		5.6 ± 1.7 a6.8 ± 3.2 a7 ± 1.6 a9.8 ± 2.4 a
N° of MVBs and dense lamellar bodies/50 μm23.6 ± 0.5 a4.4 ± 0.9 a,b6 ± 1 b9.2 ± 1.3 c
N° of CGs/10 μm2.6 ± 1.1 a2 ± 1.6 a1.6 ± 1.5 a1 ± 0.7 a
N° of microvilli/10 μm13 ± 2.7 a11.4 ± 2.8 a,b8.2± 1.5 b8.6 ± 1.1 b
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Gatti, M.; Belli, M.; De Rubeis, M.; Nottola, S.A.; Macchiarelli, G.; Tatone, C.; Di Emidio, G.; Palmerini, M.G. Concentration-Related Ultrastructural Alterations in Mouse Oocytes Following In Vitro Lindane Exposure. Appl. Sci. 2025, 15, 8320. https://doi.org/10.3390/app15158320

AMA Style

Gatti M, Belli M, De Rubeis M, Nottola SA, Macchiarelli G, Tatone C, Di Emidio G, Palmerini MG. Concentration-Related Ultrastructural Alterations in Mouse Oocytes Following In Vitro Lindane Exposure. Applied Sciences. 2025; 15(15):8320. https://doi.org/10.3390/app15158320

Chicago/Turabian Style

Gatti, Marta, Manuel Belli, Mariacarla De Rubeis, Stefania Annarita Nottola, Guido Macchiarelli, Carla Tatone, Giovanna Di Emidio, and Maria Grazia Palmerini. 2025. "Concentration-Related Ultrastructural Alterations in Mouse Oocytes Following In Vitro Lindane Exposure" Applied Sciences 15, no. 15: 8320. https://doi.org/10.3390/app15158320

APA Style

Gatti, M., Belli, M., De Rubeis, M., Nottola, S. A., Macchiarelli, G., Tatone, C., Di Emidio, G., & Palmerini, M. G. (2025). Concentration-Related Ultrastructural Alterations in Mouse Oocytes Following In Vitro Lindane Exposure. Applied Sciences, 15(15), 8320. https://doi.org/10.3390/app15158320

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