{"paper_id":"a0bd9d23-f43b-4ebf-a8e6-ccff6e67e434","body_text":"Gossypol is a naturally occurring polyphenolic compound primarily derived from the seeds, roots, and stems of  Gossypium hirsutum  L., and is abundantly present in cottonseed oil and its byproducts, such as cottonseed meal [ 1 , 2 ]. Cottonseed meal, as a high-quality protein source, is widely used in livestock and poultry production in China [ 3 , 4 ]. As a result, humans may be indirectly exposed to gossypol through the consumption of animal products—including meat, eggs, and milk—derived from animals fed with cottonseed-based feed [ 3 ].\nIn addition to its agricultural relevance, gossypol has garnered attention for its broad pharmacological activities, including antiviral, antioxidant, and antitumor effects. Clinically, it has been used to manage gynecological conditions such as uterine fibroids, endometriosis, and dysfunctional uterine bleeding [ 2 ]. Furthermore, it acts as a natural inhibitor of oncogenic proteins like APE1 and members of the BCL-2 family, showing antineoplastic activity against various cancers, including breast, ovarian, and prostate malignancies [ 1 , 2 ]. Consequently, human exposure may also occur via its oral administration or therapeutic use. Pharmacokinetic studies have shown that gossypol is primarily eliminated through the gastrointestinal tract, with minimal urinary excretion, but it exhibits uneven tissue distribution, accumulating most in the liver, followed by bile, the kidneys, and the spleen [ 4 , 5 ].\nDespite its therapeutic potential, gossypol poses significant toxicological risks. It has been shown to suppress lymphocyte proliferation, impair macrophage function, and disrupt immune responses. Additionally, it exerts neurotoxic and hepatotoxic effects, and its acute exposure has been associated with mortality in animal models [ 3 , 5 , 6 ]. Of particular concern is its reproductive toxicity. Historical epidemiological reports from China described a decade-long absence of childbirths in certain regions during the 1930s–1940s, later attributed to the dietary substitution of soybean oil with crude cottonseed oil due to economic hardship. By the 1960s, residents in several rural areas exhibited symptoms such as fatigue and burning sensations in the extremities—termed “burning fever”—followed by reproductive dysfunctions including amenorrhea in women and impotence in men, suggesting a possible link between gossypol exposure and reproductive impairment [ 6 ].\nExperimental animal studies have corroborated the reproductive toxicity of gossypol in females, demonstrating impaired folliculogenesis, reduced viable follicle counts, and increased follicular atresia, resulting in disrupted estrous cycles, embryogenesis, and pregnancy outcomes [ 7 , 8 ]. In vitro studies further confirmed its cytotoxicity toward secondary oocytes and bovine embryos [ 9 , 10 ]. However, inconsistencies in gossypol study results remain. For example, Jimenez et al. observed no significant morphological changes in preantral follicles treated with gossypol, although its high doses induced the upregulation of estrogen receptors, implying potential disruption of intra-follicular steroidogenic signaling [ 11 ]. These conflicting findings highlight the need for a systematic and multidisciplinary evaluation of gossypol’s reproductive toxicity. Additionally, it is also unclear whether ovarian function can be restored following gossypol exposure.\nAge is a critical factor influencing ovarian susceptibility to toxicants [ 12 ]. While the reproductive stage is characterized by active follicular development, reproductive senescence involves follicle depletion, hormonal imbalance, and reduced fertility [ 13 ]. The age-dependent effects of gossypol on ovarian function remain poorly understood. Moreover, previous reports of irreversible azoospermia in males following gossypol exposure raise concerns about its potential for permanent reproductive damage [ 14 ]. To address these issues, this study employed two exposure models—reproductive period and reproductive senescence—to examine stage-specific effects and assess the extent and reversibility of ovarian dysfunction caused by gossypol.\nTo ensure a systematic and evidence-based risk evaluation, this study adopted the Targeted Risk Assessment of Environmental Chemicals (TRAEC) framework. The TRAEC framework integrates four key dimensions—reliability scores, risk intensity scores, correlation scores, and integrity scores—and stratifies health risks into three levels: low (0–4), moderate (4–8), and high (8–10) [ 15 , 16 , 17 ]. By combining epidemiological data, in vivo and in vitro experiments, and mechanistic insights, the TRAEC framework offers a structured, quantitative approach for assessing reproductive risks of environmental chemicals such as gossypol, providing a robust foundation for risk mitigation in reproductive health contexts.\nThe objectives of our study are to investigate the risk of gossypol exposure on ovarian function, the differences in its effects between the reproductive and perimenopausal periods, the mechanisms underlying gossypol’s impact on ovarian function, and the reversibility of ovarian function recovery following gossypol exposure. We aim to provide experimental and theoretical foundations for the prevention and control of ovarian diseases associated with gossypol exposure.\n\nIn this study, the Targeted Risk Assessment of Environmental Chemicals (TRAEC) strategy was employed to systematically evaluate the potential risk of gossypol exposure on ovarian reserve function. This assessment was conducted in accordance with the TRAEC framework, which comprises the following sequential steps: (1) identification of the core scientific question; (2) integration of evidence derived from targeted literature searches and in-house data, encompassing epidemiological, in vivo, and in vitro studies; and (3) comprehensive evaluation of the compiled evidence across four key dimensions—study reliability (rigor of design and execution), exposure-outcome correlation (strength of association), risk intensity (alignment with the research hypothesis), and evidence integrity (completeness and consistency of evidence). These criteria were quantitatively scored to assess the overall strength of evidence and classify the level of risk associated with gossypol-induced ovarian dysfunction.\nA systematic literature search was conducted using PubMed, Web of Science, and the China National Knowledge Infrastructure (CNKI) databases, covering all publications available up to 1 April 2025. The search strategy utilized the following keywords: “gossypol” AND (“ovary” OR “oocyte” OR “granulosa cell” OR “luteal cell”). Inclusion criteria covered in vivo, in vitro, cohort, or case–control studies reporting quantitative reproductive outcomes related to gossypol exposure. Studies lacking original data or relevant endpoints were excluded. Eligible studies were scored using the TRAEC framework.\nFemale ICR mice aged 6 and 40 weeks ( n  = 40 per group) were purchased from the Animal Center of Nanjing Medical University. Female Institute of Cancer Research (ICR) mice were obtained from the Laboratory Animal Center and housed in a Specific Pathogen-Free (SPF) environment with controlled temperature (22 ± 2 °C) and humidity (55 ± 5%) and a 12 h light/dark cycle. After a 1-week acclimation, experiments were conducted following ethical approval (ethics approval number: 2406007).\nEighty female ICR mice were divided by age (6 and 40 weeks) and randomly assigned to control or gossypol groups ( n  = 20/group). Gossypol acetate (Shanghai YuanYe Bio, Shanghai, China) was given via oral gavage (20 mg/kg/day) for 30 days; controls received corn oil. Fifteen mice per group were euthanized one day post-treatment for acute toxicity evaluation. Five mice per group were monitored for 30 days to assess their recovery.\nMice were anesthetized with avertin (20 μL/g; Dalian Meilun Biotechnology, Dalian, China). Blood was collected via enucleation, and both ovaries were harvested. One ovary was fixed in 4% paraformaldehyde, paraffin-embedded, sectioned (5 μm), and H&E-stained for histology. The other ovary and serum were stored at −80 °C for molecular analyses.\nH&E-stained serial ovarian sections were examined using a Nikon Ni-E microscope. Follicles were categorized as follows: primordial follicles—oocytes surrounded by a single layer of flattened granulosa cells; primary follicles—oocytes enclosed by a single layer of cuboidal granulosa cells; secondary follicles—two or more granulosa cell layers and zona pellucida; antral follicles—presence of an antral cavity and cumulus mass; and atretic follicles—degenerated oocyte, disrupted granulosa layers, or detachment. Every third section was analyzed; only follicles with visible nucleoli were counted to prevent duplication.\nSerum AMH and FSH levels were measured using ELISA kits (Jianglai Bio, Shanghai, China). Assays were performed per manufacturer instructions, including serial dilution, standard/sample addition to 96-well plates, incubation with HRP-conjugated antibodies, substrate reaction, and OD detection at 450 nm using a BioTek Synergy2 reader (BioTek Instruments, Inc., Winooski, VT, USA). Hormone concentrations were calculated from standard curves (R 2  ≥ 0.98). All samples were assayed in duplicate.\nA 50 mM gossypol stock was prepared by dissolving 0.029 g of gossypol acetate in 1 mL of DMSO. Its working concentrations (0, 30, 60, and 120 μM) were made by adding 0, 0.6, 1.2, and 2.4 μL of the stock solution to 1 mL of M16 medium (Sigma-Aldrich, St. Louis, MO, USA), pre-equilibrated at 37 °C in 5% CO 2 . Ovaries from six 6-week-old ICR mice were collected after cervical dislocation. Germinal vesicle (GV)-stage oocytes were isolated under a stereomicroscope (Olympus SZX16, Tokyo, Japan) and cultured (40–50 per group) in media with respective gossypol concentrations. GVBD and PBE rates were assessed at 4 h and 14 h, respectively.\nGV-stage oocytes were collected from five 6-week-old ICR mice and treated with 0, 30, 60, or 120 μM gossypol to assess cytoskeletal and mitochondrial alterations.\nSpindle assembly and Chromosome alignment: After 9 h of culture, oocytes were fixed (4% paraformaldehyde), permeabilized (0.5% Triton X-100), and blocked. Spindles were stained with anti-α-tubulin antibody (Thermo Fisher Scientific, Waltham, MA, USA), and nuclei were stained with Hoechst 33,342 (KeyGen BioTECH, Nanjing, China). Images were captured using a Zeiss LSM700 confocal microscope.\nMitochondrial distribution: After 8 h, oocytes were incubated with MitoTracker Red CMXRos (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h, then fixed and imaged to assess mitochondrial localization.\nKGN cells (from Prof. Ran Huo, Nanjing Medical University) were cultured in DMEM (Gibco, Carlsbad, CA, USA) with 10% FBS (NEWZERUM, Christchurch, New Zealand), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 °C and 5% CO 2 .\nA 40 μg/mL gossypol stock was prepared (0.002 g gossypol in 100 μL DMSO, diluted in 50 mL medium). Its working concentrations (0, 5, 10, and 20 μg/mL) were applied to cells in 24-well plates for 24 h. Proliferation was assessed using an EdU kit (Beyotime, Shanghai, China) following standard protocols: EdU incubation (2 h), fixation, permeabilization, click reaction, Hoechst 33,342 counterstaining (KeyGen BioTECH, Nanjing, China), and imaging on a Nikon Ti2-U microscope. EdU-positive nuclei were quantified to calculate proliferation rates.\nTUNEL assays were performed under the same gossypol treatment concentrations and duration as the EdU assay. After 24-h treatment, apoptosis was detected using the TUNEL Cell Apoptosis Kit (Beyotime, China). Cells were fixed in 4% paraformaldehyde, permeabilized with PBS, incubated with TUNEL reaction mix, and imaged via fluorescence microscopy. The proportion of TUNEL-positive cells was used as an index of apoptosis.\nOvaries from three control and three gossypol-treated 6-week-old mice were analyzed by data-independent acquisition (DIA) mass spectrometry (Majorbio Bio-Pharm, Shanghai, China). Total proteins were extracted and trypsin-digested, and the obtained peptides were analyzed using LC-MS/MS with an Astral high-resolution mass spectrometer (400–1200  m / z , DIA mode). Data were processed via Spectronaut using project-specific or library-free workflows. Peptide FDR was controlled at <1%. Differentially expressed proteins (DEPs) were defined as proteins with a fold change of ≥1.5 or ≤0.67 with  p  < 0.05. GO and KEGG analyses were performed for functional annotation.\nData were analyzed using GraphPad Prism 10.0 (GraphPad Software, San Diego, CA, USA) and presented as the mean ± SD. Student’s  t -test was used for two-group comparisons; one-way ANOVA with Tukey’s post hoc test for multiple groups. Mann–Whitney U test was applied to non-parametric data.  p  < 0.05 was considered statistically significant.\n\nGossypol has recently gained attention for its potent antitumor properties, making it a promising candidate for cancer therapy. Initially developed as a male contraceptive, its clinical use was discontinued due to concerns about irreversible infertility. However, data on its reproductive toxicity in female germ cells remain scarce. Thus, this study aims to address the following critical question: does gossypol exposure impair ovarian reserve function in females?\nFollowing a predefined strategy, 250 articles related to gossypol exposure and ovarian function were retrieved from PubMed, Web of Science, and CNKI. After removing 63 duplicates, 187 articles were screened based on inclusion and exclusion criteria. Full-text review led to the exclusion of 10 additional articles due to insufficient data or methodological flaws. Ultimately, 36 studies were included in the TRAEC-based systematic risk assessment ( Figure 1 ).\nKey characteristics of the 36 included studies are summarized in  Table 1  and  Table 2 .\nAlthough gossypol has been studied for gynecological conditions such as endometriosis, uterine fibroids, and abnormal bleeding [ 7 ], no epidemiological studies specifically examined its impact on ovarian reserve. Among the included studies, two studies combined in vivo and in vitro experiments, resulting in 38 independent datasets: 17 in vivo animal studies and 21 in vitro cellular studies.\nSeventeen in vivo studies were identified, utilizing various animal models including mice, rats, cows, ewes, hamsters, bats, and ducks. Specifically, six studies used rats; four used cows; two used mice; two used ewes; and one each used hamsters, bats, and ducks ( Table 1 ). All studies evaluated the structural and functional effects of gossypol on ovarian tissue.\nA total of 21 in vitro studies were included, employing diverse cell and tissue types ( Table 2 ), including granulosa cells ( n  = 6); luteal cells ( n  = 6); oocytes ( n  = 4); whole ovarian tissue ( n  = 2); and one study each on theca cells, ovarian cells, isolated follicles, and oocyte–cumulus complexes. Collectively, these studies provide a comprehensive view of gossypol’s impact on ovarian cell morphology, function, hormone synthesis, and developmental competence, forming a robust evidence base for risk assessment.\nTo evaluate the effects of gossypol on ovarian reserve at different reproductive stages, female mice aged 6 weeks (young group) and 40 weeks (aged group) were orally administered gossypol daily for 30 days. Post-exposure, 6-week-old mice exhibited visible clinical symptoms, including sparse, coarse fur and hemorrhagic signs (petechiae and ecchymosis) on the tail, which were absent in controls ( Figure 2 A). Both age groups showed significant body weight reduction compared with their respective controls (6-week-old: control 35.90 ± 3.19 g vs. gossypol 30.26 ± 0.91 g; 40-week-old: control 49.17 ± 1.20 g vs. gossypol 46.88 ± 1.25 g) ( Figure 2 B). Ovarian volume and ovary-to-body weight ratios were significantly decreased following treatment, with more pronounced changes in the young group ( p  < 0.0001) and significant reductions in the aged group ( p  < 0.001) ( Figure 2 C,E). Histopathological analysis using H&E staining revealed marked disruption of ovarian architecture in gossypol-treated mice ( Figure 2 D,F). Ovaries from control animals displayed intact histology, with follicles at all developmental stages, abundant primordial follicles, and minimal atresia. In contrast, gossypol-treated ovaries exhibited reduced total follicle counts, depletion of primordial and primary follicles, increased atresia, and partially collapsed antral follicles with oocyte degeneration. Quantitative follicle analysis confirmed significant reductions in the numbers of primordial ( p  < 0.01), primary ( p  < 0.01), and secondary follicles ( p  < 0.05) in 6-week-old mice. Antral follicle counts also declined ( p  < 0.05), while atretic follicles significantly increased ( p  < 0.01). In the 40-week-old group, similar trends were observed, with decreased numbers of primordial and primary follicles ( p  < 0.05), and a 5-fold increase in the atretic follicle ratio ( p  < 0.01) compared with controls. To further assess endocrine function, serum levels of anti-Müllerian hormone (AMH) and follicle-stimulating hormone (FSH) were measured ( Figure 2 G,H). In both age groups, AMH and FSH levels showed a mild upward trend after gossypol treatment; however, these changes were not statistically significant ( p  > 0.05).\nGerminal vesicle breakdown (GVBD) and first polar body extrusion (PBE) rates were assessed after 4 and 14 h of in vitro culture, respectively ( Figure 3 A). At 4 h, control oocytes showed a GVBD rate of 90.39 ± 3.91%, whereas gossypol treatment (30, 60, and 120 μM) caused a significant, dose-dependent reduction to 76.07 ± 2.95%, 73.46 ± 3.76%, and 48.22 ± 5.68% ( p  < 0.05). At 14 h, PBE rates declined from 65.88 ± 8.44% in controls to 33.67 ± 3.22% (30 μM,  p  < 0.001), 22.62 ± 2.28% (60 μM,  p  < 0.0001), and 2.86 ± 1.17% (120 μM,  p  < 0.001), indicating substantial inhibition of maturation. Based on these findings, 60 μM was the chosen gossypol concentration for subsequent mechanistic studies.\nAfter 9 h of culture, control oocytes exhibited barrel-shaped spindles with correct chromosome alignment ( Figure 3 B). In contrast, 60 μM of gossypol significantly increased spindle abnormalities and misaligned chromosomes (71.6 ± 10.41% vs. 13.33 ± 11.55%,  p  < 0.01). Additionally, MitoTracker staining showed a dramatic reduction in mitochondrial activity (6.4 ± 1.43% vs. 28.93 ± 5.41% in controls,  p  < 0.0001) ( Figure 3 C). This suggests that gossypol disrupts both spindle formation and mitochondrial function, thereby impairing cytoplasmic maturation and oocyte competence.\nEdU and TUNEL assays demonstrated that gossypol impairs proliferation and induces apoptosis in human granulosa-like tumor (KGN) cells. EdU incorporation decreased with increasing gossypol concentrations: 29.41 ± 7.42% at 5 μg/L ( p  < 0.01), 21.02 ± 0.68% at 10 μg/L, and 16.48 ± 3.06% at 20 μg/L (both  p  < 0.001) ( Figure 3 D). TUNEL staining revealed a dose-dependent increase in apoptosis: 18.50 ± 14.04% (5 μg/L,  p  > 0.05), 55.12 ± 2.70% (10 μg/L,  p  < 0.001), and 91.06 ± 2.23% (20 μg/L,  p  < 0.0001) compared with the control (11.46 ± 9.75%) ( Figure 3 E). Together, these results indicate that gossypol exerts direct cytotoxic effects on ovarian granulosa cells by inhibiting proliferation and promoting apoptosis.\nIn this study, we applied a multi-evidence-based Targeted Risk Assessment of Environmental Chemicals (TRAEC) strategy to systematically evaluate the potential reproductive toxicity of gossypol on ovarian reserve function. The assessment focused on four core dimensions: reliability scores, risk intensity scores, correlation scores, and integrity scores. Each study was independently scored by two reviewers, with discrepancies resolved via discussion involving a third reviewer. This two-tiered review process enhances objectivity and scientific validity. Reliability was rated on a 1–10 scale ( Figure 4 A). Correlation scores are summarized in  Figure 4 B. Risk intensity (0–1 scale) indicates the degree to which findings support the study hypothesis ( Figure 4 C). Evidence count reflects the total number of studies across all dimensions. Integrity was adjusted to reflect the exclusion of epidemiological studies by redistributing weight between in vivo and in vitro evidence. The overall TRAEC-derived risk assessment yielded a composite score of 4.68 ( Figure 4 D), indicating moderate risk (score range: 4–8). No protective effects were observed.\nIn summary, this integrative TRAEC analysis classifies gossypol exposure as a moderate reproductive risk, due to its significant evidence of ovarian reserve impairment. These findings underscore the need for cautious use of gossypol in clinical settings and warrant further targeted investigations.\nTo elucidate molecular mechanisms underlying gossypol-induced ovarian dysfunction, we performed a comprehensive proteomic comparison between ovaries from 6-week-old female mice treated with gossypol and age-matched controls ( Figure 5 ). Mass spectrometry detected 8328 proteins, with reliable quantification of 8121 proteins across all samples. Sample correlation analysis demonstrated high intra-group reproducibility (R 2  > 0.98) and clear inter-group discrimination (R 2  < 0.5), consistent with principal component analysis (PCA) results ( Figure 5 A,B). Differential expression analysis revealed 151 significantly altered proteins following gossypol treatment, with 43 upregulated and 108 downregulated proteins (fold change ≥ 2 or ≤0.5;  p  < 0.05) ( Figure 5 C). KEGG pathway enrichment indicated that downregulated proteins primarily participate in cell adhesion, steroid hormone biosynthesis, and antigen processing/presentation pathways ( Figure 5 D), suggesting that gossypol disrupts ovarian endocrine, immune, and structural functions. Conversely, upregulated proteins were enriched in pathways related to protein digestion/absorption and basal cell carcinoma ( Figure 5 E), possibly reflecting compensatory stress responses.  Figure 5 F highlights the five most significantly downregulated proteins, associated with immune regulation, cell adhesion, and metabolism. Notably, glutathione synthetase (GSS)—a key enzyme in glutathione, cysteine, and methionine metabolism—suggests oxidative stress as a contributor to ovarian toxicity.  Figure 5 G presents the top five upregulated proteins, including the mitochondria-localized NAD(P)H pyrophosphatase NUDT13 and fatty acid-binding protein 3 (FABP3), the latter linked to PPAR signaling, indicating disruptions in lipid metabolism and energy homeostasis.\nOverall, proteomic profiling indicates that gossypol exposure induces extensive alterations in critical signaling pathways—steroidogenesis, immune regulation, mitochondrial metabolism, and cell adhesion—thereby impairing ovarian cellular function and depleting ovarian reserve capacity.\nTo evaluate the potential reversibility of gossypol-induced ovarian damage, subsets of 6-week-old and 40-week-old female mice underwent a 30-day recovery period following cessation of gossypol treatment. Throughout recovery, body weights in treated mice remained slightly lower than their age-matched controls (6 weeks: control 46.38 ± 2.39 g vs. gossypol 42.14 ± 0.83 g; 40 weeks: control 56.57 ± 1.37 g vs. gossypol 54.91 ± 1.64 g), with no further decline, suggesting systemic stabilization ( Figure 6 A). After one month of withdrawal, ovarian volume and ovary-to-body weight ratios did not differ significantly between treated and control mice in either age group ( p  > 0.05), indicating partial morphological recovery ( Figure 6 B,E). Histological evaluation showed modest increases in primordial and primary follicles and a reduction in atretic follicles in both groups compared with post-treatment levels. However, none of these follicular metrics returned to control baselines, and differences remained statistically non-significant ( p  > 0.05), indicating incomplete structural restoration ( Figure 6 C,F). Endocrine assessments revealed slight post-withdrawal increases in serum AMH and FSH, but these did not reach control levels or statistical significance ( Figure 6 G). Consequently, current data are insufficient to conclude that gossypol causes sustained endocrine disruption, warranting further investigation.\nWithin a one-month recovery period, gossypol-induced ovarian damage appears only partially reversible. Although ovarian structure and follicular composition showed modest improvement, endocrine and functional recovery was not significant. These results suggest that gossypol may exert lasting effects on ovarian reserve, and further research is needed to determine the extent and reversibility of its reproductive endocrine impact.\n\nThis study systematically evaluated the effects of gossypol exposure on ovarian reserve function and its reversibility in female mice, elucidating its potential reproductive toxicity and endocrine-disrupting effects. We quantitatively assessed the reproductive toxicity of gossypol utilizing the TRAEC strategy that integrates multiple lines of evidence from epidemiological, in vivo, and in vitro studies.\nDespite numerous studies reporting the reproductive effects of gossypol, results have been inconsistent [ 8 , 9 , 11 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 ], and the overall reproductive risk has not been clearly quantified. We adopted the TRAEC strategy to address this gap, offering a comprehensive, systematic, and quantitative risk assessment. Based on 36 eligible studies, we determined a moderate reproductive risk level (risk score: 4.68) associated with gossypol exposure. This integrative approach overcomes the limitations of relying on a single type of evidence, providing a more accurate toxicity profile for environmental chemicals. Notably, no epidemiological studies in human populations were identified, despite gossypol acetate’s clinical use for treating uterine fibroids and endometriosis [ 51 , 52 ], and its potential as an anticancer agent [ 1 , 53 , 54 , 55 , 56 ]. These findings highlight the need for future population-based investigations to determine the potential reproductive impact of gossypol in humans.\nOur findings demonstrate that gossypol exposure significantly impairs ovarian function in female mice. Macroscopically, treated mice exhibited reduced body weight and smaller ovarian volume, suggesting inhibited overall growth and ovarian development. Previous studies support this observation, with Luz et al. (2018) reporting follicular atresia rates of 65–88% in rats, mice, and goats following seven days of gossypol treatment, and another study observing up to 79.4% atretic follicles in sheep [ 19 ]. Our study also showed that the number of growing follicles at all stages decreased after gossypol exposure, while the number of atretic follicles increased. In isolated oocyte experiments, 60 μM gossypol disrupted spindle formation and mitochondrial membrane potential, leading to a 63.7% decrease in polar body extrusion and increased oxidative stress [ 34 , 36 ]. These studies are consistent with our research findings. Granulosa cell studies indicate that gossypol inhibits proliferation and dysregulates steroidogenic enzymes (CYP11A1, HSD3B1, CYP17A1), reducing estradiol-17β and progesterone production, while leaving LH receptor expression unchanged [ 35 , 37 , 39 ]. Additionally, estrogen receptor α expression was altered [ 11 ]. Additionally, we also found that gossypol exposure induces apoptosis in granulosa cells. Together, these results suggest that gossypol impairs ovarian function through compound mechanisms involving follicle depletion, hormone disruption, and oocyte maturation failure.\nPrevious studies have not reported on the effects of gossypol on ovarian function in different age groups. Our research findings indicate that gossypol exposure led to notable structural damage within the ovarian tissue and significantly depleted the follicular reserve in both young and aged mice. Notably, the ovarian damage was considerably more severe in the 6-week-old group, suggesting that ovaries during the reproductive age may be more susceptible to gossypol-induced toxicity. This highlights a critical high-risk window that warrants further investigation to better understand the underlying mechanisms, potentially leading to the development of preventive measures or targeted therapies to mitigate the adverse effects of gossypol on ovarian health. Advances in omics technologies have deepened our understanding of gossypol’s ovarian effects.\nAdditionally, the development of omics technologies assists in elucidating the mechanisms of ovarian damage caused by gossypol. Metabolomic and redox proteomic studies in SKOV3 granulosa cells [ 56 ] and RNA-seq in primary granulosa cells [ 35 ] have been conducted, although whole-ovary omics remains unexplored. The cellular complexity of ovarian tissue—including oocytes, granulosa, and immune, stromal, and endothelial cells [ 57 ]—warrants comprehensive proteomic analysis. Our proteomic profiling revealed that gossypol disrupted pathways related to protein metabolism and carcinogenesis (upregulated), while downregulating pathways involved in cell adhesion, steroidogenesis, and antigen presentation. Notably, immune-related proteins such as OCIA2, H-2 class II antigen α-chain, and GALNT1 were significantly reduced [ 58 , 59 , 60 ], implicating immune dysregulation in ovarian impairment—an established factor in primary ovarian insufficiency [ 61 , 62 ]. Additionally, proteins involved in mitochondrial function and metabolism (NUDT13, FABP3) were altered, and glutathione synthetase—a key enzyme in redox homeostasis—was downregulated [ 63 , 64 , 65 ]. These findings align with prior evidence of gossypol’s impact on germ cell metabolism and redox balance [ 34 , 36 ].\nWe synthesized the existing literature on gossypol’s effects on female fertility and identified multiple mechanisms through which it impairs ovarian function. Gossypol disrupts oocyte development by impairing both nuclear and cytoplasmic processes—including spindle assembly and mitochondrial function [ 34 , 36 ]. It may also disturb the balance between granulosa and theca cell proliferation and apoptosis. Additionally, gossypol interferes with endocrine regulation by altering sex hormone production and disrupting estrogen receptor expression [ 35 , 37 , 39 ]. Moreover, it impairs cellular energy metabolism, increases ROS generation and lipid peroxidation, damages cellular ultrastructure, and induces apoptosis—particularly targeting mitochondria [ 34 , 35 , 36 ]. Our findings further suggest an immunoregulatory component to its action, potentially involving disruption of the hypothalamic–pituitary–ovarian (HPO) axis, leading to follicular atresia and endocrine dysfunction.\nHistorically, gossypol was evaluated as a male contraceptive [ 66 ], but concerns about irreversible fertility effects limited its use [ 14 , 67 ]. A multicenter study of 151 men treated for up to 16 weeks revealed that, one year post-treatment, only 10 of 24 participants in the 10 mg/day group exhibited recovered sperm counts, while 8 of 43 remained azoospermic [ 14 ]. A mouse study by Wang et al. found that, although short-term gossypol exposure caused reproductive and renal toxicity, these effects reversed following withdrawal [ 68 ]. In contrast, our study—which spanned over one complete mouse follicular cycle (17–19 days) [ 69 ] during both exposure and recovery—demonstrated only partial restoration of follicle numbers and hormone levels after one month, indicating that gossypol-induced ovarian damage may be partially irreversible. This may stem from structural ovarian damage (e.g., follicle depletion and increased apoptosis) that is not readily repairable and sustained hormonal imbalances hinting at long-term endocrine disturbance. These findings underscore the importance of fertility protection in women receiving gossypol acetate for gynecological or oncological conditions.\nHowever, our study still has the following limitations: First, as this study was conducted using a murine model, its applicability to humans may be limited. Future studies should monitor fertility outcomes in women exposed to gossypol to evaluate both short- and long-term reproductive effects. Second, although proteomic analysis revealed multiple disrupted pathways, the interactions among these pathways and their specific molecular mechanisms require further investigation. Advanced approaches such as gene-knockout or overexpression models may help elucidate the roles of key regulatory molecules. Third, the recovery phase was limited to one month; extending this period could yield a more comprehensive understanding of the reversibility of gossypol-induced ovarian impairment.\n\nThis study applied an innovative environmental chemical risk assessment framework (i.e., TRAEC) to evaluate the effects of gossypol exposure on ovarian reserve by integrating published evidence with novel experimental data. The results demonstrate that gossypol poses a moderate reproductive risk, as it reduces ovarian reserve in female mice, impairs oocyte maturation, and disrupts pathways involved in hormone production and antigen processing. In vitro assays further confirmed that gossypol inhibits granulosa cell proliferation and induces apoptosis. Although partial recovery was observed one month after exposure cessation, residual functional impairment and endocrine alterations persisted, indicating potentially irreversible reproductive effects. Future studies should focus on human-based investigations to clarify gossypol’s long-term impact on ovarian health and explore alternative compounds—such as dextrorotary (+) -gossypol—that may offer therapeutic benefits without compromising fertility [ 70 ].","source_license":"CC-BY-4.0","license_restricted":false}