Silent Legacy I: Paternal Phthalate Exposure Shifts Androgen–Estrogen Receptor Balance and Induces Transgenerational Reproductive Disruption, Signs of Autism, Feminization and Homosexuality in Mice

Silent Legacy I: Paternal Phthalate Exposure Shifts Androgen–Estrogen Receptor Balance and Induces Transgenerational Reproductive Disruption, Signs of Autism, Feminization and Homosexuality in Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Silent Legacy I: Paternal Phthalate Exposure Shifts Androgen–Estrogen Receptor Balance and Induces Transgenerational Reproductive Disruption, Signs of Autism, Feminization and Homosexuality in Mice Reda Ali, Heba Aboulqasem, Dalia Elzahraa Mostafa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8600132/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The increasing prevalence of reproductive and neurodevelopmental disorders has raised concern over endocrine-disrupting chemicals (EDCs). Phthalates, including di-butyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP), are widely used industrial chemicals that disrupt hormonal regulation and impair male fertility. This study examined the effects of paternal prepubertal exposure to DBP and DEHP on endocrine function, receptor expression, and sexual behavior in Swiss albino mice. Sixty-three F₀ males (21 days old) were divided into control and treated groups receiving DBP & DEHP at 100, 200 & 400 mg/kg body weight for 15 days, followed by recovery. Hormonal assays for testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were performed, and estrogen receptor alpha (ERα) and androgen receptor (AR) expression were evaluated immunohistochemically. Low dose exposed males were bred to generate F₁ and subsequently F₂ offspring. Paternal phthalate exposure reduced testosterone, increased LH and FSH, upregulated ERα, and attenuated AR expression, with alterations persisting into adulthood. Behavioral assessments revealed impaired sexual performance, delayed mating, reduced motivation, feminization, homosexuality, autism like traits, and circadian rhythm disruption in F₀ males, effects persisting in F₁ offspring. These findings demonstrate that paternal phthalate exposure induces lasting endocrine, receptor, and behavioral disturbances with multigenerational consequences, underscoring the need for safety evaluation. Health sciences/Endocrinology Biological sciences/Physiology DEHP circadian rhythm digging LH Free testosterone ejaculation latency Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction The rising incidence of reproductive and neurodevelopmental disorders has raised concerns about environmental toxicants, particularly endocrine-disrupting chemicals (EDCs), during critical developmental periods. A central hypothesis is that these disorders share a developmental origin linked to early adverse exposures 1 . The Endocrine Society estimates that of the over 80,000 human synthesized chemicals currently in use globally, only 1% have been assessed for safety, but thousands could act as EDCs 2 . Growing awareness of the reproductive and developmental toxicity of synthetic chemicals has therefore prompted renewed scrutiny of phthalates, especially regarding in utero exposure and its long-term effects 1 . Phthalates comprise up to half of total mass of certain plastic products, underscoring their extensive use as plasticizers. Among these compounds, di (2-ethylhexyl) phthalate (DEHP) is particularly prominent, accounting for roughly 25% of worldwide phthalate production. DEHP is a low-volatility liquid widely used as a plasticizer in flexible polyvinyl chloride products 3 . These materials are commonly found in consumer products including synthetic leather, waterproof clothing, footwear, upholstery, floor tiles, furnishing, industrial tubing, cables, tablecloths, shower curtains, food packaging, children's toys, and wide range of medical devices 4 . It is utilized to enhance flexibility and durability 5 . According to EU classification, DEHP is recognized as a reproductive toxicant, designated as Reprotoxic Category 2, with the following risk statements. R61 “May cause harm to the unborn child” and R60 – “May impair fertility” 6 . Recently, growing attention has been directed toward the presence of phthalates in perfumes, where they are intentionally incorporated as solvents and fragrance fixatives. Primary phthalates used in cosmetic products include di-butyl phthalate (DBP), which is used as a stabilizer in nail polishes, hair sprays, and perfumes 7 . Structurally, phthalate esters are defined by a diester configuration, featuring a benzene dicarboxylic acid backbone conjugated to two ester side chains. These compounds are not chemically bound to the plastic matrix, making it easy for them to leach into the surrounding environment including air, water, food, and soil resulting in widespread human and ecological exposure 5 . Human exposure to phthalates occurs via ingestion, inhalation, dermal absorption, and transplacental transfer during pregnancy 8 . DBP has been detected in consumer items in concentrations ranging from 444.6 to 1671.1 μg/mL 9 . The developmental period is particularly sensitive to EDC exposure, as it is characterized by intense cellular differentiation and hormonal regulation. Numerous studies have demonstrated that phthalates disrupt reproductive processes by mimicking or antagonizing natural hormones, leading to abnormalities such as testicular atrophy, impaired spermatogenesis, and reduced fertility. DEHP has been shown to alter the expression of genes involved in testis development and steroidogenesis, with Sertoli cells being primary targets for phthalate-induced damage. Furthermore, immature animals appear more susceptible to these effects than adults, with gonadal damage following DEHP exposure in young rats 10 . In addition to reproductive toxicity, phthalates have increasingly been implicated in sexual, neurobehavioral and psychiatric disturbances. The global rise in neuropsychiatric disorders may be partially explained by the pervasive use of EDCs such as phthalates, bisphenols, and vinclozolin 11 . DBP is a male-specific reproductive toxicant 5 , while DEHP is linked to reduced play in boys, elevated metabolites in autistic children, and anxiety- and depression-like behaviors in animals 10 . One of the most concerning findings about EDCs is their ability to produce intergenerational and transgenerational effects. Phthalates can influence germline epigenetic programming, leading to inherited reproductive and behavioral abnormalities across multiple generations. These include reduced sperm counts, altered stress hormone levels, impaired social behaviors, and disrupted brain development in descendants never directly exposed to the chemical 10,12 . Mechanistically, EDCs act through complex pathways, including genomic and non-genomic nuclear receptor activation, ion channels, nonsteroidal receptors, and transcriptional regulators 12 . These chemicals bind to receptors meant for naturally produced (endogenous) hormones that are essential to everything from growth to reproduction 2 . Given the alarming rise in phthalate consumption such as a 30% increase in phthalic anhydride use over the two past decades 13 and the mounting evidence of their reproductive and behavioral toxicity, further studies are needed to clarify the scope and mechanisms of their effects. Owing to their extensive and prevalent application in a wide range of consumer and industrial products, DEHP and DBP were selected as representative compounds for investigation in the present study, aiming to examine behavioral consequences of prepubertal exposure­­­­ in male mice, with a specific focus on the potential for transgenerational transmission of these effects through the male germ line (sperm). By comparing the impacts on both reproductive potential and sexual behavior across generations, this research seeks to contribute to the understanding of how phthalate exposure during development may have lasting implications for and fertility. 2. Results 2.1. Sex hormones level 2.1.1. At 36 days of age FSH levels significantly increased in all treatments compared to control, exerting the highest effect by the median dose (200 mg/kg/bw). The lowest (100 mg/kg/bw) and highest (400 mg/kg/bw) doses were lesser effective than the median one, respectively, yet remaining significantly elevated above control levels ( p < 0.0001, F 6, 14 = 221.5, ƞ p 2 =0.989576; Fig. 2a). LH levels were significantly elevated in DBP-treated groups compared to the control, with the highest observed level in DBP (200 mg/kg/bw), followed by DBP (100) and DBP (400). In contrast, DEHP-treated groups exhibited a moderate dose dependent increase in LH levels. DEHP (400) showed a significant elevation compared to control ( p 0.05; Fig. 2b). A significant reduction in serum free testosterone levels was observed in all treated groups compared to the control ( p < 0.0001, F 6, 14 = 2166, ƞ p 2 =0.998924). Both DBP and DEHP induced a dose-dependent decline, with the highest dose (400 mg/kg/bw) causing the most pronounced decrease, showing insignificant difference among DBP and DEHP ( p > 0.05; Fig. 2c). 2.1.2. At 50 days of age FSH levels showed a general decline compared to the elevated levels observed at 36 days, indicating improvement in hormonal regulation. In DBP groups, FSH levels were significantly increased only at the 100 mg/kg/bw dose, while DBP 200 and 400 returned to near control levels. In contrast, DEHP groups exhibited a dose dependent increase in FSH: DEHP 100 showed insignificant change, DEHP 200 showed a significant elevation, and DEHP 400 displayed a highly significant increase. Overall, FSH levels were improved by day 50, particularly in DBP 200 and 400 groups, while higher doses of DEHP continued to elevate FSH, indicating a delayed recovery at high exposure ( p < 0.0001, F 6, 14 = 37.21, ƞ p 2 =0.940993; Fig. 3a). LH levels showed a general decline compared to the elevated levels observed at 36 days, indicating partial hormonal recovery. However, LH remained significantly elevated in all treated groups compared to control. In DBP groups, LH levels increased in a dose dependent manner. In DEHP groups, a more pronounced dose dependent increase was observed with the highest dose (400 mg/kg/bw) showing the highest LH level. Overall, while LH levels at day 50 were lower than that at day 36, especially in DBP groups, they remained significantly elevated particularly in DEHP 400 suggesting incomplete but notable hormonal improvement over time ( p < 0.0001, F 6, 14 = 29.42, ƞ p 2 =0.926517; Fig. 3b). Free testosterone levels showed partial recovery compared to the lower levels observed at 36 days. However, levels in all DBP and DEHP groups remained significantly lower than control. Among DBP-treated groups, DBP (100 mg/kg/bw) showed the highest improvement, while DBP 200 remained the lowest. DBP 400 showed moderate recovery. In DEHP treated groups, free testosterone levels remained consistently low, with DEHP 200 showing the least recovery, while DEHP 100 and 400 showed similar levels. Overall, free testosterone showed some improvement by day 50, particularly in DBP 100 and 400 groups, but hormone levels did not return to control values ( p < 0.0001, F 6, 14 = 903.6, ƞ p 2 =0.997424; Fig. 3c). 2.2. Immunohistochemical expression of estrogen (ER α ) and androgen (AR) receptors 2.2.1. At 36 days of age 2.2.1.1. Estrogen receptors (ERα) In control testes, ER α expression was largely restricted to Leydig cells cytoplasm, with minimal staining in peritubular myoid layer (PTL) (Fig. 4a). In treated groups, ER α expression was markedly increased: DBP 200 showed the most extensive and intense staining across Leydig, Sertoli, spermatogonia and PTL (Fig. 4c). At the highest dose (400 mg/kg) of DBP and DEHP, staining remained strong in Sertoli cells, spermatogonia, and the PTL, but Leydig cells were not detected (Fig. 4d & g). The remaining treated groups displayed ER α expression mainly in Leydig cells and PTL , but with markedly increased intensity compared to control (Fig. 4 b,e&f). These qualitative observations were confirmed by quantitative analysis of optical density (OD), which showed a statistically significant increase in ERα expression in all treated groups compared to control ( p=0.0007 , F 6, 63 = 12.61, ƞ p 2 =0.545651; Fig. 4h). Importantly, ERα expression was consistently higher in all DBP treated groups than in the corresponding DEHP treated groups across all doses. Among these, the DBP (200) dose induced the most pronounced upregulation of ERα expression. Although DEHP treatment at all doses (100, 200, and 400) resulted in significantly higher ERα expression than the control group, no statistically significant differences were observed among the DEHP doses themselves ( p>0.05 ). 2.2.1.2. Androgen receptors (AR) In control testes, AR expression was highly pronounced in the nuclei of Sertoli cells, Leydig cells cytoplasm and PTL (Fig. 5a). In treated groups, AR expression was inhibited in the seminiferous tubules and was primarily confined to Leydig cells and PTL (Fig. 5 b-e). However, in the DEHP 200 and 400 groups, Leydig cells were absent, with AR staining confined to the PTL (Fig. 5 f&g) . Statistically, all treated groups showed a significant decrease in AR expression compared to control group ( p< 0.0001 , F 6, 63 = 10.12, ƞ p 2 = 0.490786; Fig. 5h). However, the lower dose of DBP (100) as well as the low and high doses of DEHP (100 and 400) displayed the lowest AR expression among all treated groups. 2.2.2. At 70 days of age 2.2.2.1. Estrogen receptors (ERα) ERα expression in control testes was confined to Leydig cells cytoplasm, with no detectable staining in the seminiferous tubules (Fig. 6a) . In treated groups, ERα expression persisted in Leydig cells cytoplasm and was also observed in some Sertoli cells (Fig. 4 b-g) , with DBP 200 additionally showing expression in spermatogonia and spermatids (Fig. 6c) , indicating continued upregulation in these cells. Statistical analysis of OD revealed that ERα expression was significantly increased only in the DBP 100 group compared with the control ( p= 0.0159 , F 6, 63 = 2.856, ƞ p ² = 0.214; Fig. 6h). Although other treated groups showed elevated ERα levels, these increases were statistically insignificant ( p>0.05 ). 2.2.2.2. Androgen receptors (AR) AR expression in control testes showed no detectable staining within the seminiferous tubules and was confined to Leydig cells cytoplasm (Fig. 7a). In treated testes, abnormal AR expression was observed, with some staining present in Sertoli nuclei (Fig. 7 b,c,e&f); however, the highest dose (400) of both DBP and DEHP did not exhibit any AR expression within the seminiferous tubules, while expression in Leydig cells cytoplasm was maintained (Fig. 7 d&g). Statistically, analysis of OD revealed a persistent significant decrease in AR expression in most treated groups compared to control ( p=0.0038 , F 6, 63 = 3.615, ƞ p 2 = 0.256111; Fig. 7h). The lowest AR expression among treated groups was observed in the highest and lowest doses of DBP (100 and 400) and the intermediate dose of DEHP (200). 2.3. Effects of phthalate exposure on sexual behavior across generations 2.3.1. F ₀ Generation 2.3.1.1. Mount latency & frequency Although mount latency tended to increase in both phthalate treated groups relative to controls, particularly in the DEHP group, these differences were not statistically significant ( p= 0.0563, F 2, 6 = 4.829, ƞ p 2 =0.616809; Fig. 8a). Mount frequency were significantly affected. Both DBP and DEHP groups exhibited a significant increase in mount frequency compared to control group ( p 0.05; Fig. 8b). 2.3.1.2. Intromission latency Intromission latency was significantly increased in the DBP and DEHP groups compared to control ( p= 0.0130, F 2, 15 = 5.889, ƞ p 2 =0.439839), indicating a delay in initiating successful copulatory behavior. No significant difference was observed among DBP and DEHP groups ( P > 0.05; Fig. 8c). 2.3.1.3. Ejaculation latency Ejaculation latency was significantly reduced in DBP and DEHP groups compared to control ( p= 0.0035, F 2, 15 = 8.466, ƞ p 2 =0.530252), with insignificant difference between DBP and DEHP groups ( P > 0.05; Fig. 8d). 2.3.1.4. Post ejaculatory interval Post ejaculatory interval was significantly reduced in DEHP group compared to control and DBP groups ( p= 0.001 , F 2, 15 = 11.46, ƞ p 2 =0.60443). Insignificant difference was observed among control and DBP groups ( p > 0.05; Fig. 8e). 2.3.1.5. Inter-mating Interval Inter-mating interval was significantly increased in DBP and DEHP groups compared to control ( p < 0.05). DBP group showed the longest interval, which was significantly longer than both control and DEHP groups. Additionally, DEHP group had a significantly longer interval than control but was significantly shorter than DBP group ( p= 0.0401, F 2, 6 = 5.763, ƞ p 2 =0.657651; Fig. 8f). 2.3.2. F 1 Generation 2.3.2.1. Mount Latency Mount latency showed insignificant increase across treated groups compared to control, with more pronounced elevations observed in the TC(B/E) and TT(B) groups ( p= 0.5443, F 6, 14 = 0.8637, ƞ p 2 = 0.270157; Fig. 9a). 2.3.2.2. Mount Frequency Mount frequency was significantly increased in TC (B/E) and TT (B/E) groups compared to control group ( p < 0.0001, F 6, 35 = 182.3, ƞ p 2 =0.968994). TT (B) group exhibited the highest mount frequency among all other groups. TC (E) group also showed a significantly higher frequency than control, CT (B/E) groups. No significant difference was observed among control and both CT (B/E) groups ( p > 0.05; Fig. 9b). 2.3.2.3. Intromission Latency Intromission latency was significantly increased in TC (B/E) and TT (E) groups compared to control ( P < 0.0001, F 6, 35 = 10, ƞ p 2 =0.631579). TT (E) group showed the longest latency among all other groups. In contrast, insignificant differences were observed among control and CT (B/E) and TT (B) groups ( p > 0.05; Fig. 9c). 2.3.2.4. Ejaculation Latency Ejaculation latency was significantly altered across treated groups compared to control. CT (B/E) groups showed significantly longer ejaculation latency than control. In contrast, TC (B/E) and TT (B/E) groups exhibited significantly shorter ejaculation latency than control. Among them, TT (B) being the most reduced ( p < 0.0001 , F 6, 35 = 116.1, ƞ p 2 =0.95216; Fig. 9d). 2.3.2.5. Post ejaculatory Interval Post ejaculatory interval was significantly reduced in all treated groups compared to control. TT (B) and TC (E) groups showed the most pronounced reductions, with values significantly lower than other groups ( p < 0.0001 , F 6, 35 = 52.37, ƞ p 2 =0.899777; Fig. 9e). 2.3.2.6. Inter-mating Interval Insignificant differences were observed among control and all treated groups. Among all groups, TC (E) exhibited the highest inter-mating interval, while other exposed groups showed intermediate changes compared to control ( p= 0.6809, F 6, 14 = 0.6627, ƞ p 2 =0.221192; Fig. 9f). 2.3.3. Sexual motivation Under normal physiological conditions, male mice exhibit mating behavior approximately every two to three days depending on the strain; a pattern consistently observed in the control group. Video recordings confirmed that control males successfully mated with both females and made multiple escape attempts, ultimately succeeding, which reflects normal sexual motivation and power. The escaped male gained access to a separately housed female outside the primary experimental setup, and that female subsequently became pregnant. Control males employed two distinct escape strategies driven by sexual motivation: (1) repeatedly jumping to cling to the wire-mesh ceiling and gnawing the wax seal (videos 1&2), and (2) persistently enlarging the water inlet until it was wide enough to escape (video 3). In contrast, treated males displayed a marked delay in initiating mating behavior, with intervals extending up to 13 days. Notably, some males in TT (B) group failed to initiate mating altogether during the observation period, and exhibited no escape attempts, indicating a potential reduction in sexual drive and behavioral responsiveness. 2.3.4. Circadian rhythm A clear disruption of circadian rhythm was observed in most treated groups across both generations, marked by a temporal shift in mating activity from the evening, as seen in controls, to the early morning. In control males, mating activity peaked around 9.00 p.m. (video 4), whereas treated groups exhibited a marked shift, with copulatory behavior occurring predominantly between 4.00 and 7.00 a.m. (videos 5-8). This alteration was accompanied by an inverted sleep–wake cycle, with both mating and general locomotor activities occurring predominantly during morning rather than during nocturnal phase. 2.3.5. Digging & jumping In both F₀ (parental) and F₁ (offspring) generations, a range of abnormal behaviors were consistently observed in the treated groups, indicating possible transgenerational effects of phthalate exposure. One of the most frequent behaviors was excessive and repetitive digging , particularly in sawdust bedding. This behavior was more pronounced in DEHP treated animals across both generations (videos 9&10). Along with abnormal repeated jumping and hyperactivity ( videos 11&12) , behaviors that were absent or minimal in control group. These signs of altered activity patterns were consistent across both generations in DBP treated animals. 2.3.6. Homosexuality Notably, female–female sexual behaviors, such as mounting and mating attempts, were observed exclusively in (TT) groups and were absent in controls. This behavior was particularly pronounced in the F₁ generation (videos 13&14). It is worth noting that the male that did not exhibit any mating attempts was housed with females that displayed female–female mounting behavior. 2.3.7. Feminization Under normal conditions, when a group of females is housed with a single male, females usually sleep close to each other while the male rests separately (video 15). In the present experiment, however, some DBP treated males were observed sleeping in close contact with females, which may reflect feminization related behavioral alterations (video 16), an atypical behavior which was not noted in control males, which predominantly slept alone. Although this behavior was not frequently observed, its presence represents a deviation from typical male resting patterns. Moreover, males in the F 1 generation TT (B) displayed no mounting attempts, further indicating the persistence of feminized behavior in offspring derived from DBP exposed males. 3. Discussion The present study demonstrates that paternal exposure to phthalates in the F₀ generation induced persistent endocrine disruption, characterized by reduced testosterone, compensatory elevations in LH and FSH, ERα upregulation, and AR attenuation. These hormonal alterations were accompanied by sexual and neurobehavioral impairments, including delayed mating, reduced sexual motivation, feminization, and autism-like behaviors, which persisted into the F₁ generation. The observed reductions in free testosterone, along with compensatory elevations in LH and FSH, are indicative of potential primary testicular failure (hypogonadism), wherein the testes fail to adequately produce sperm or synthesize testosterone. Consistent with previous studies who established that testosterone, the principal androgen produced by Leydig cells, is essential for spermatogenesis and normal male sexual behavior, and that disruption of androgen signaling can impair copulatory efficiency and sexual motivation 14,15 . Moreover, exposure to phthalate metabolites has been linked to adult-onset hypogonadism and endocrine disruption, affecting gonadotropin balance and testicular function 16,17 . Specifically previous studies 18 have reported phthalate induced suppression of key steroidogenic genes and proteins, suggesting that the observed severe testosterone deficiency in the present study may be a consequence of phthalate induced disruption of the genes responsible for its biosynthesis. Testosterone exerts its biological effects primarily through the androgen receptor, which is expressed in Leydig cells, Sertoli cells, peritubular myoid cells, and germ cells 19 . Disruption of AR function, whether through genetic mutations or receptor loss, results in androgen insensitivity, testicular feminization, and impaired spermatogenesis, as demonstrated in AR knockout models 20 . Reduced AR expression in Sertoli and peritubular myoid cells has also been reported in cases of non-obstructive azoospermia, highlighting the essential role of AR signaling in maintaining spermatogenic integrity 21 . Phthalates are well established EDCs with pronounced anti-androgenic properties. Experimental studies have shown that phthalates inhibit AR activity, act as antagonists in reporter gene assays, and interfere with receptor dimerization and downstream transcriptional signaling, ultimately suppressing androgen dependent gene expression in Sertoli and germ cells 22 . Also, recent study reporting that elevated urinary DBP metabolite levels are associated with altered male reproductive hormones 23 , supporting the endocrine-disrupting effects of phthalates. The markedly reduced testosterone levels observed in the present study further confirm the suppression of androgenic signaling induced by DBP and DEHP exposure. In the present study, attenuation or absence of AR expression in treated testes is likely attributable to the anti-androgenic actions of phthalates. Interestingly, while AR expression in control testes at 70 days of age was relatively low, consistent with the attainment of endocrine stability at sexual maturity, the re-emergence of AR expression in a subset of Sertoli cells in treated animals may represent a compensatory response to earlier androgen suppression. Such delayed or atypical receptor re-expression has been interpreted as a cellular attempt to restore diminished androgenic signaling 24 . However, at the highest phthalate dose (400 mg/kg/bw), toxic effects may override these compensatory mechanisms, consistent with established dose dependent toxicity principles 25 . Despite attenuated AR expression, spermatogenesis persisted in treated testes, coinciding with enhanced ERα expression. Estrogens play a critical role in male reproductive physiology through modulation of the hypothalamic–pituitary–gonadal axis and local regulation of spermatogenesis within the testis 26 . Both ERα and ERβ are expressed in Leydig cells, Sertoli cells, peritubular myoid cells, and germ cells, with species-specific distribution patterns 27 . Estrogen signaling through these receptors regulates germ cell differentiation, survival, and apoptosis, thereby coordinating spermatogenic progression 27,26 . In the current study, ERα overexpression was observed in treated testicular sections, particularly in the DBP treated groups. Alterations in ERα expression have been associated with pathological conditions of the male gonad. Increased ERα expression in Leydig cells has been reported in cryptorchid testes, suggesting that excessive estrogen signaling contributes to impaired testicular function 28 . Similarly, in utero DBP exposure has been shown to increase ERα expression while reducing ERβ and AR expression, indicating a shift toward estrogen dominant signaling within the testicular microenvironment 29 . DEHP exposure has also been reported to alter testicular gene expression profiles related to hormonal signaling and receptor regulation 30 . Also, latest evidence indicates that that phthalates exhibited AR antagonist effect based on non-AR dimerization and agonistic effect of ERα 31 . In agreement with these reports, the present study suggests that the observed estrogen overexpression may be explained by two converging mechanisms: a direct estrogenic action of phthalates and an indirect effect mediated by phthalate induced androgen deficiency, which removes androgen dependent inhibitory control over estrogen signaling. Together, these mechanisms promote ERα overexpression and estrogen-dominant signaling. In the present study, phthalate exposure, directly in F₀ and indirectly in F₁ generations, resulted in significant alterations in male sexual behavior, particularly affecting timing and structure of mating activity. Increased mount and intromission latencies indicated a delay in the initiation of effective copulatory behavior, while elevated mount frequencies observed in the treated groups may reflect heightened, yet disorganized or inefficient, sexual attempts. These findings are consistent with earlier studies reported that phthalate exposure induced increased intromission latency and larger number of intromissions to achieve ejaculation 32 , indicating that phthalates adversely affected the efficiency of copulatory behavior. The significant reduction in ejaculation latency in treated groups, alongside a shortened post ejaculatory interval, suggests an alteration in the refractory phase of sexual behavior, potentially impairing normal sexual pacing. This reduction may reflect a disruption in neuroendocrine regulation of sexual satiety or recovery mechanisms. Several studies have reported that phthalate exposure induced anxiety like behaviors in rodents 33 . Consistent with these findings, behavioral assessments in the present study revealed that treated mice displayed clear indicators of anxiety. Previous experiments have shown that anxiety in male mice is associated with a significant reduction in ejaculation latency 34 . Therefore, it is plausible that the ejaculation latency reduction observed in the present phthalate groups may be mediated by phthalate induced anxiety. In another study, phthalate exposure likely determines epigenetic alterations in programming of sex brain differentiation and regulation of testicular steroidogenesis, leading to long-term reproductive and behavioral disorders 35 . In agreement with those studies, it is suggested that phthalate-induced anxiety, neuroendocrine disruption, and epigenetic reprogramming likely act together to shorten ejaculation latency and impair sexual pacing. The present observations revealed a prolonged inter-mating interval reaching delays of up to 13 days in certain treated groups TC (E). Control male mice typically resume mating within hours to a few days depending on the strain; for instance, C57BL/6 males average approximately 4 days, while DBA/2 males may remate within an hour 36 . A prolonged interval of this magnitude therefore signals a marked disruption in reproductive function, likely reflecting a combination of reduced sexual motivation, neurobehavioral impairment, and physiological dysfunction. Such alterations are consistent with previous findings that EDCs such as DBP and DEHP interfere with androgen signaling, suppress testosterone synthesis, and disrupt central neuroendocrine circuits regulating sexual behavior 37 . Recently, it was found that many male rats exposed to phthalates in utero and during lactation were sexually inactive in the presence of receptive females, and this inactivity was not associated with morphological abnormalities, suggesting a disturbance in sexual brain differentiation 35 . Notably, insignificant differences were found between DBP and DEHP groups for most parameters, suggesting that both phthalates exert comparable disruptive effects on timing and coordination of sexual performance. Sexual motivation in rodents is a well-established model for studying reward-driven behavior. The escape behavior observed in the current control male mice, leaving the cage to access a receptive female indicates strong sexual motivation rather than exploratory activity. Operant paradigms show that males will repeatedly nose-poke or lever-press to gain access to a mate, reflecting high effort expenditure for sexual reward 38 . Similarly, the electrified grid barrier model, first described in early behavioral studies 39 , demonstrated that males would endure aversive electrical stimulation to approach a female, underscoring that sexual motivation can override discomfort an observation consistent with current findings. In contrast, this escape response was absent in all phthalate males, providing strong evidence that phthalate exposure reduces sexual motivation. Reduced androgen receptor expression and testosterone levels likely underlie this loss of sexual motivation. In the present study, female–female sexual behaviors were observed exclusively in the F ₁ generation. A similar phenomenon has been reported, with the emergence of homosexual-type behaviors in male rats prenatally exposed to low doses of DBP 35 . In their study, 18-month-old males exhibited testicular and accessory gland atrophy, reduced testosterone, increased Leydig cell adenomas, and impaired sexual performance. These effects were linked to prenatal DBP-induced epigenetic changes disrupting brain sexual differentiation and testicular steroidogenesis. By analogy, the female–female sexual interactions documented in the current research may similarly reflect phthalate-induced alterations in neuroendocrine pathways governing sexual behavior. Such disruptions could originate from changes in the organizational effects of sex steroids during critical developmental periods, ultimately leading to atypical sexual behaviors and potential reproductive dysfunction later in life. Estrogen signaling plays a key role in shaping social affiliation and sexual behavior, and excessive ERα activation during critical developmental windows has been shown to bias neural circuits toward feminized behavioral outputs 40,27 . In the present study, treated males exhibited increased affiliative proximity to females during rest, a clearly feminized social behavior alongside ERα expression in the testicular tissue, suggests a relative estrogenic shift resulting from altered estrogen–androgen signaling. Disruption of this hormonal balance is known to interfere with male sexual differentiation and behavioral organization, and altered estrogen signaling has been associated with changes in male typical behaviors following prenatal phthalate exposure in humans 40 . Also, phthalate exposure has been linked to feminization related behavioral alterations and increased anxiety like behavior 33 . Notably, in the present study, DBP exposure induced a more pronounced ERα expression than DEHP, supporting previous evidence that DBP preferentially enhances estrogen receptor mediated signaling while suppressing androgenic pathways 29,31 . This molecular difference was reflected behaviorally, as DBP treated males exhibited more overt feminized behaviors, whereas such behaviors were less evident in DEHP exposed groups. Consistent with this interpretation , endocrine signaling is compelled to shift toward one of two hormonal pathways. Consequently as discussed above, the loss of androgenic signaling leaves estrogen signaling as the dominant and functionally main pathway, promoting ERα overexpression and estrogen dominant signaling, which may underlie the observed feminized behavior in male mice. In contrast, DEHP exposure was more closely associated with neurobehavioral abnormalities, including repetitive and anxiety like behaviors 41,42,1 , suggesting preferential disruption of neurodevelopmental pathways. Importantly, the persistence of altered sexual and social behaviors in both the F₀ and F₁ generations may be attributed to disruption of the pubertal organizational period, which has been described as a second critical window during which gonadal hormones permanently modify neural processing in brain regions regulating sexual behavior 14 . Collectively, these findings indicate that phthalate exposure induces estrogen receptor overexpression alongside attenuation of androgenic signaling in the male gonad, thereby contributing to disrupted sexual differentiation and feminized behavioral traits. This highlights the critical importance of balanced estrogen–androgen signaling in maintaining normal male reproductive and behavioral phenotypes and underscores the potential long-term consequences of phthalate exposure on male endocrine and neurobehavioral health. Overall, these findings highlight a serious concern, suggesting that phthalate exposure could contribute to the emergence of effeminized male traits in human and may represent one of several environmental factors potentially linked to the increasing incidence of homosexuality in certain communities. DBP was first demonstrated to interfere with the normal functioning of circadian rhythm circuits which regulate rhythmic expression of numerous genes 17,43 . Disruption of circadian gene expression has been implicated in the pathogenesis of various diseases, including cancer 44 . These findings are consistent with the current study, in which disruption of biological clock regulation was observed in DBP exposed groups manifested as altered rest-activity cycles and abnormal sleep-wake behaviors, thereby suggesting that DBP induced disruption of circadian gene expression and may contribute to broader physiological and behavioral disturbances. Collectively, sexual behavior impairments, circadian rhythm disturbances, abnormal jumping and feminization were predominantly observed in animals directly exposed to DBP or derived from DBP treated parents, suggesting a compound-specific disruption of reproductive neuroendocrine pathways. In contrast, neurobehavioral abnormalities including repetitive digging were primarily associated with DEHP exposure or parental exposure to DEHP, implicating distinct epigenetic or neurodevelopmental mechanisms in mediating these effects. Recent studies emphasize that phthalate effects are strongly influenced by dose, timing of exposure, and sex, supporting evidence that these chemicals can drive multigenerational and transgenerational alterations in development and reproduction 12 . These findings are relevant for understanding how even low dose exposures to environmental chemicals can affect not just the direct offspring but also future generations. It is important to consider the effects of environmental exposures on neurodevelopment, as numerous studies have demonstrated their significant impact on autism spectrum disorder (ASD) related behaviors. ASD is characterized by repetitive behaviors, social deficits, communication difficulties, stereotyped patterns of behavior, and heightened anxiety 45 . Prenatal phthalate exposure can disrupt synaptic function, impair learning and memory, and alter circadian rhythms, ultimately increasing the risk of ASD development in children 46 . Similarly, the concept of “environmentally vulnerable physiology,” emphasizing how environmental factors can exacerbate neurodevelopmental vulnerabilities, has been highlighted 47 . Previous findings using various DEHP exposure models have demonstrated alterations in anxiety-like and social interaction behaviors, particularly in males 42 . Recent evidence suggests that phthalate metabolites might interfere with tyrosine and tryptophan metabolism and consequently affect neurobehavioral development 1 . Male mice exposed to 200 mg/kg DEHP displayed elevated digging behavior and decreased self-grooming, both nonsocial behavioral alterations frequently associated with autism-like phenotypes. Consistent with these results, DEHP exposure during gestational and early postnatal development has been linked to increased anxiety in pubertal male offspring, as evidenced by more time spent in the closed arms of the elevated plus maze 33 . Mechanistically, DEHP regulates gene expression and induces neuronal degeneration in the hippocampus, a brain region critical for learning and memory 41 , which may explain the emergence of repetitive digging or jumping behaviors observed in DEHP treated mice in the present study. Collectively, these findings of repetitive behaviors, impaired locomotion, disrupted circadian rhythms, reduced sexual activity, heightened anxiety, and potential cognitive deficits in phthalate-exposed males parallel ASD-like phenotypes in humans. These results suggest that early-life phthalate exposure disrupts typical neurobehavioral trajectories in a sex-specific manner and may contribute to autism risk in children, either through direct exposure during infancy (e.g., pacifiers and feeding bottles) or through potential transgenerational effects associated with parental plastic use. Importantly, transgenerational impacts were evident in F₂ generation. This observation aligns with the proposed concept that when both parents carry the same environmentally or epigenetically influenced trait, the likelihood of its expression in offspring is significantly increased 48 . Consistent with this principle, the present study found that TT group derived from both a treated male and a treated female displayed the most pronounced alterations across behavioral and physiological parameters, supporting the hypothesis of cumulative or synergistic parental effects mediated through heritable epigenetic modifications. The TC group, in which only the male was treated, showed intermediate effects, while the CT group, where the male was unexposed, exhibited the least abnormalities. These patterns highlight the critical role of parental exposure, particularly male exposure in shaping offspring outcomes and further suggest that phthalate-induced disruptions can be amplified when both parents are affected. 4. Materials and Methods The current study was achieved in accordance with the Egyptian laws and University guidelines for animal care. The National Ethical Committee of Assiut University, Faculty of Science Research Ethics Committee (FSREC), Egypt, has approved all the procedures in the present work with the approval number: 01-2025-0017. All procedures involving animals were conducted in accordance with the ARRIVE guidelines. 4.1. Chemicals DEHP and DBP, with 98% purity, were purchased from SRL (India). The chemicals were merged into corn oil (used as a vehicle) before administration and were stored at room temperature. 4.2. Experimental Animals A total of 81 albino mice (Swiss strain) designated as the F₀ generation were obtained from the animal house of the Faculty of Science, Al-Azhar University, Assiut. The experiment was initiated with the 63 immature males aged 21 days, weighing 8–12 g on average, which were randomly assigned to seven groups: a control group, the remaining six groups were subdivided into three groups for each of DEHP and DBP. These groups were orally administered, once daily, a volume of 100 µl of corn oil carrying the three doses (100, 200 & 400 mg/kg bw) for fifteen consecutive days. Three mice from each group were euthanized at ages of 36, 50 & 70 days by cervical dislocation without prior anesthesia, in accordance with institutional and international guidelines for the care and use of laboratory animals. At ages of 36 and 50 days, blood samples were collected to measure follicle-stimulating hormone (FSH), luteinizing hormone (LH) and free testosterone (FT). At ages of 36 and 70 days, estrogen receptor alpha (ERα) and androgen receptor (AR) expression was assessed by immunohistochemical investigation. These doses for both DBP & DEHP were chosen based on previous studies 49,50 respectively. Animals were provided food and water ad libitum under standard laboratory conditions, maintained at a temperature of 23 ± 2 °C and a 12-hour light/dark cycle. To comply with research ethics, the number of animals used was minimized, and stress on the mice was also reduced by avoiding unnecessary handling and by ensuring that only a single observer was present in the room throughout the experiment. Experiments were carried out in strict compliance with the ethics prepared by INSA and (WHO/UNESCO). Following a 15-day recovery period after treatment cessation and upon reaching sexual maturity, mating commenced. 4.3. Hormonal measurements Blood was collected from the retro-orbital sinus, centrifuged at 5000 rpm for 20 min; serum was separated and stored at –20 °C until analysis. Serum concentrations of free testosterone, FSH, and LH were measured using immunoassay kits (Free Testosterone, FSH, and LH; Roche Diagnostics, Switzerland) on a Cobas e411 immunoassay analyzer, following the manufacturer’s instructions. 4.4. Immunohistochemical investigation The immunohistochemical investigation was carried out on formalin-fixed testis collected at ages of 36 and 70 days. The specimens were processed, embedded in paraffin, and the tissue sections were cut at 4μm. The sections were treated with 10 mL MoL Tris buffer and 1 mL MoL ethylene-diamine tetra-acetic acid, pH 9.0 for 20 min at 90 °C. The block of endogenous peroxidase was done by incubation the sections with 3% H2O2, followed by preincubation overnight at 4 °C in 1% bovine serum albumin in PBS. The sections were stained for 30 min at 37 °C, using the following antibodies that showed reactivity in mice species: a rabbit polyclonal anti-AR (1:200; ABclonal Technology, Wuhan, China; Cat. No. A16200), and a rabbit polyclonal anti-ERα (1:200, Affinity Biosciences, Wuhan, China; Cat. No. AF6058), according to the method described previously 51 . Sections were counterstained with haematoxylin, and analyzed using an Honor 400 mobile camera. In parallel, tissue specimens, in whom the primary antibodies were omitted and replaced with buffer, served as negative controls. 4.5. Breeding the F0, F1 The F₀ generation comprised three experimental groups: control, DBP, and DEHP. In each group, three immature males were randomly selected from animals exposed to a dose of 100 mg/kg DBP or DEHP, and from the corresponding control group. To produce the F₁ generation, each male was mated with two untreated virgin females weighing 21–25 g, resulting in six females per group (18 in total). Thus, the parental generation (F₀) comprised 27 animals. For the F 2 generation, three mating schemes were designed for each treatment group (DBP & DEHP). Control male × indirect treated female CT (B/E), indirect treated male × control female TC (B/E), and indirect treated male × indirect treated female TT (B/E), where (B) indicates DBP treated and (E) indicates DEHP treated parents. In this notation, the first letter always refers to the male and the second to the female (Fig. 1). Each scheme was replicated three times, yielding nine pairs per group in addition to three replicates of the control × control group. In total, 21 mating pairs (42 animals) were used to generate the F₂ offspring. Mating of the F 0 generation was conducted during the period from (22-4-2024) to (12-5-2024) in the DBP lineage and from (12-5-2024) to (31-5-2024) in the DEHP lineage. Likewise, mating of the F 1 generation occurred from (5-7-2024) to (24-7-2024) in the DBP lineage and from (27-7-2024) to (15-8-2024) in the DEHP lineage. 4.6. For behavioral assessment Each male was individually housed in a glass cage with wire mesh for ventilation under controlled light/dark conditions. A video camera was activated for one hour, after which two females were introduced into the cage and cohabitated with the male for 20 days. Body weights of both males and females were recorded daily during this period. Sexual behavior was evaluated by measuring parameters including mount latency & frequency, intromission latency, ejaculation latency, post-ejaculatory interval and Inter-mating Interval according to 52 . Any abnormal or atypical behavior was also noted. Behavioral observations were recorded using a HiLook 2 MP surveillance camera (resolution. 1920×1080 pixels, frame rate. 30 fps), at the Zoology & Entomology Department, Faculty of Science, Assiut University. All experimental procedures applied to the F₀ generation—including video recording, and behavioral assessments—were consistently replicated for the F₁ generation. All procedures involving animals were conducted in accordance with the ARRIVE guidelines. 4.7. Statistical analysis Data were estimated as mean ± SE. Hormonal data and optical density (OD) were analyzed statistically using column statistics and one-way analysis of variance (one-way ANOVA), and the test of Newman-Keuls multiple comparison test as a post test. For sexual behavior assessments, to exclude potential female related effects, a paired t-test was applied to compare measurements obtained from the first and second females mated with the same male. No significant differences were detected between females for any of the assessed parameters. Accordingly, data from both females were considered as biological replicates representing the same male, and group comparisons were subsequently performed using one-way analysis of variance (one-way ANOVA). These analyses were carried out using Prism software for windows, version 5.0 (Graph pad software Inc., San Diego, California, USA) and Excel (Microsoft office 10). Analysis of OD was carried out using the software Image J (the JAVA SE Runtime Enviroment, version 6). Declarations Author contributions statement Reda A. A. suggested the point, read the manuscript, contributed in interpretation of the results, and revised the manuscript. Dalia Elzahraa F. M. contributed to reviewing the paper. Heba E. A. achieved the practical part and contributed to preparing, writing and reviewing the paper. All authors read and approved the final manuscript. Data Availability All data generated or analyzed during this study are included in this published article. Additional Information Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. ORCID for authors Reda. A. Ali. ORCID https://orcid.org/0000-0002-6716-9789 Telephone No. 00201011423969 Dalia Elzahraa F. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8600132","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":577057070,"identity":"0fa200c4-2e46-4f6e-9292-2e6fbe0489c3","order_by":0,"name":"Reda Ali","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYHACA4YPMAbRWhhnkKyFmYckLebtzRs/29QczmNgb94mwVBRS1iLzJljxdI5xw4XM/AcK5NgOHOcsBYJiRwD6Ry2w4kNEjlmEoxtx4jQIv/G+LfFP6AW+TdALf+I0SLBYybN2AayhQeopaGGCC08aWWWvX3piW08acUWCccOEKGF/fDmGz++WSf2sx/eeONDTR1hLXDABiISGA6ToAUKSLFlFIyCUTAKRgoAANKZNQYGczqaAAAAAElFTkSuQmCC","orcid":"","institution":"Assiut University","correspondingAuthor":true,"prefix":"","firstName":"Reda","middleName":"","lastName":"Ali","suffix":""},{"id":577057071,"identity":"4edf491c-bcca-4013-b120-d1b07cecc701","order_by":1,"name":"Heba Aboulqasem","email":"","orcid":"","institution":"Assiut University","correspondingAuthor":false,"prefix":"","firstName":"Heba","middleName":"","lastName":"Aboulqasem","suffix":""},{"id":577057072,"identity":"d5068100-4ad4-4894-983c-726c712f7bc5","order_by":2,"name":"Dalia Elzahraa Mostafa","email":"","orcid":"","institution":"Assiut University","correspondingAuthor":false,"prefix":"","firstName":"Dalia","middleName":"Elzahraa","lastName":"Mostafa","suffix":""}],"badges":[],"createdAt":"2026-01-14 09:38:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8600132/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8600132/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100754108,"identity":"b159b14c-9a4d-43ae-8670-b425dbccf9e5","added_by":"auto","created_at":"2026-01-21 05:51:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6689367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of paternal phthalate exposure and its impact on F0 sires as well as F1 and F2 offspring, illustrating the timeline required to complete the experiment. The diagram also illustrates the different mating combinations used to generate the second (F₂) generation, which included. (1) indirect treated male × indirect treated female (T T), (2) indirect treated male × control female (T C), and (3) control male × indirect treated female (C T).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8600132/v1/06263069cd3f624e8df3fca7.png"},{"id":100754035,"identity":"6c22799f-fdfa-4c9b-ac3a-c2169661ba52","added_by":"auto","created_at":"2026-01-21 05:50:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3902971,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum hormone levels at 36 days in control, DBP and DEHP exposed groups (100, 200, 400 mg/kg). \u003c/strong\u003e(a) FSH (mIU/ml). (b) LH (mIU/ml). (c) Free testosterone (pg/ml). \u003cem\u003eDifferent letters indicate statistically significant differences between groups.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8600132/v1/ce4460f23c5c79f9b223fea8.png"},{"id":100754100,"identity":"87561866-7a9f-4cc5-98e7-c3d118b929be","added_by":"auto","created_at":"2026-01-21 05:51:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3729544,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum hormone levels at 50 days in control, DBP and DEHP exposed groups (100, 200, 400 mg/kg).\u003c/strong\u003e (a) FSH (mIU/ml). (b) LH (mIU/ml). (c) Free testosterone (pg/ml). \u003cem\u003eDifferent letters indicate statistically significant differences between groups.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8600132/v1/fbd4cc15ba3f1dc34f2ddaaa.png"},{"id":100754046,"identity":"8e7c739d-4e97-4d18-825e-89309c914100","added_by":"auto","created_at":"2026-01-21 05:50:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74350175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical detection of ERα in testicular tissue at 36 days of age. \u003c/strong\u003eRepresentative micrographs: (a) Control, (b) DBP 100 mg/kg, (c) DBP 200 mg/kg, (d) DBP 400 mg/kg, (e) DEHP 100 mg/kg, (f) DEHP 200 mg/kg, (g) DEHP 400 mg/kg. \u003cstrong\u003eLeydig cells (thick arrows) and spermatogonia (SG). \u003c/strong\u003eX400. (h) Optical density (OD) measurements. \u003cem\u003eDifferent letters (a, b, c) denote significant differences between groups.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8600132/v1/61c151a39013fd9acefc3ea3.png"},{"id":100754044,"identity":"5698819e-69b3-4bc1-b016-4efd216a13cd","added_by":"auto","created_at":"2026-01-21 05:50:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":70873752,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical detection of AR in testicular tissue at 36 days of age. \u003c/strong\u003eRepresentative micrographs: (a) Control, (b) DBP 100 mg/kg, (c) DBP 200 mg/kg, (d) DBP 400 mg/kg, (e) DEHP 100 mg/kg, (f) DEHP 200 mg/kg, (g) DEHP 400 mg/kg\u003cstrong\u003e. Leydig cells (thick arrows) and Sertoli cells (arrowheads).\u003c/strong\u003eX400. (h) Optical density (OD) measurements. \u003cem\u003eDifferent letters (a, b, c) denote significant differences between groups.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8600132/v1/c4c1e5c58ee1ce65dc1acbd8.png"},{"id":100754037,"identity":"966e119b-9dd1-408d-82b2-368fa30d66cb","added_by":"auto","created_at":"2026-01-21 05:50:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":71974189,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical detection of ERα in testicular tissue at 70 days of age. \u003c/strong\u003eRepresentative micrographs: (a) Control, (b) DBP 100 mg/kg, (c) DBP 200 mg/kg, (d) DBP 400 mg/kg, (e) DEHP 100 mg/kg, (f) DEHP 200 mg/kg, (g) DEHP 400 mg/kg. \u003cstrong\u003eLeydig cells (thick arrows), Sertoli cells (arrowheads) spermatogonia (SG) and spermatids (ST).\u003c/strong\u003e X400. (h) Optical density (OD) measurements. \u003cem\u003eDifferent letters (a, b, c) denote significant differences between groups.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-8600132/v1/45f43393c7199153d05a2aae.png"},{"id":100754113,"identity":"07668845-a4bc-4d3f-87d5-0f78880af08c","added_by":"auto","created_at":"2026-01-21 05:51:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":70460565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical detection of AR in testicular tissue at 70 days of age.\u003c/strong\u003eRepresentative micrographs: (a) Control, (b) DBP 100 mg/kg, (c) DBP 200 mg/kg, (d) DBP 400 mg/kg, (e) DEHP 100 mg/kg, (f) DEHP 200 mg/kg, (g) DEHP 400 mg/kg. \u003cstrong\u003eLeydig cells (thick arrows) and Sertoli cells (arrowheads).\u003c/strong\u003eX400. (h) Optical density (OD) measurements. \u003cem\u003eDifferent letters (a, b, c) denote significant differences between groups.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-8600132/v1/f96f8a13f53a98adb1fe47f6.png"},{"id":100754098,"identity":"b90cd7c7-42df-4b5d-806f-d90597f547f9","added_by":"auto","created_at":"2026-01-21 05:51:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5173375,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSexual behavior parameters in F₀ males across control, DBP, and DEHP exposed groups.\u003c/strong\u003e(a) Mount latency. (b) Mount frequency. (c) Intromission latency. (d) Ejaculation latency. (e) Post-ejaculatory interval. (f) Inter-mating interval. \u003cem\u003eDifferent letters (a, b, c) denote significant differences between groups.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-8600132/v1/c909dc74d62550857dc0e398.png"},{"id":100754031,"identity":"da2efbb5-0ad7-4f1d-abf2-b9005e16fe79","added_by":"auto","created_at":"2026-01-21 05:50:43","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":6828374,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSexual behavior parameters in F₁ males across control and treatment groups [CT(B), TC(B), TT(B), CT(E), TC(E), TT(E)].\u003c/strong\u003e (a) Mount latency (days). (b) Mount frequency. (c) Intromission latency. (d) Ejaculation latency. (e) Post-ejaculatory interval. (f) Inter-mating interval. \u003cem\u003eDifferent letters (a–f) denote statistically significant differences between groups.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.9.png","url":"https://assets-eu.researchsquare.com/files/rs-8600132/v1/6fb85f99ac83c5643b5c5cf2.png"},{"id":101751711,"identity":"eb474285-14ef-4b01-a3fb-462c15d416da","added_by":"auto","created_at":"2026-02-03 10:22:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":149451513,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8600132/v1/4c2d990e-764a-41fe-b3a8-f82905cb0b7f.pdf"},{"id":100754027,"identity":"587f38bb-9611-46f7-99d4-de5717665f9e","added_by":"auto","created_at":"2026-01-21 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05:50:47","extension":"docx","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":13727,"visible":true,"origin":"","legend":"","description":"","filename":"Highlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-8600132/v1/e98413e155e9cfaa662fbdb9.docx"},{"id":100754081,"identity":"ab9de653-e197-4492-ae3c-1acff35bba85","added_by":"auto","created_at":"2026-01-21 05:51:22","extension":"tif","order_by":18,"title":"","display":"","copyAsset":false,"role":"supplement","size":11252744,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-8600132/v1/f9e4ca3d67ac6e635c7eda28.tif"},{"id":100754026,"identity":"e100db9b-4b70-4d02-9d16-2ad8bcb2a770","added_by":"auto","created_at":"2026-01-21 05:50:40","extension":"docx","order_by":19,"title":"","display":"","copyAsset":false,"role":"supplement","size":14349,"visible":true,"origin":"","legend":"","description":"","filename":"VideoLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-8600132/v1/38ebebc035a052226b364c9f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Silent Legacy I: Paternal Phthalate Exposure Shifts Androgen–Estrogen Receptor Balance and Induces Transgenerational Reproductive Disruption, Signs of Autism, Feminization and Homosexuality in Mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe rising incidence of reproductive and neurodevelopmental disorders has raised concerns about environmental toxicants, particularly endocrine-disrupting chemicals (EDCs), during critical developmental periods. A central hypothesis is that these disorders share a developmental origin linked to early adverse exposures\u003csup\u003e1\u003c/sup\u003e.\u0026nbsp;The Endocrine Society estimates that of\u0026nbsp;the over 80,000 human synthesized chemicals currently in use globally, only 1% have been assessed for safety, but thousands could act as EDCs\u003csup\u003e2\u003c/sup\u003e. Growing awareness of the reproductive and developmental toxicity of synthetic chemicals has therefore prompted renewed scrutiny of phthalates, especially regarding \u003cem\u003ein utero\u003c/em\u003e exposure and its long-term effects\u003csup\u003e1\u003c/sup\u003e. Phthalates comprise up to half of total mass of certain plastic products, underscoring their extensive use as plasticizers. Among these compounds, di (2-ethylhexyl) phthalate (DEHP) is particularly prominent, accounting for roughly 25% of worldwide phthalate production. DEHP is a low-volatility liquid widely used as a plasticizer in flexible polyvinyl chloride products\u003csup\u003e3\u003c/sup\u003e. These materials are commonly found in consumer products including synthetic leather, waterproof clothing, footwear, upholstery, floor tiles, furnishing, industrial tubing, cables, tablecloths, shower curtains, food packaging, children\u0026apos;s toys, and wide range of medical devices\u003csup\u003e4\u003c/sup\u003e. It is utilized to enhance flexibility and durability\u003csup\u003e5\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccording to EU classification, DEHP is recognized as a reproductive toxicant, designated as Reprotoxic Category 2, with the following risk statements. R61 \u0026ldquo;May cause harm to the unborn child\u0026rdquo; and R60 \u0026ndash; \u0026ldquo;May impair fertility\u0026rdquo;\u003csup\u003e6\u003c/sup\u003e. Recently, growing attention has been directed toward the presence of phthalates in perfumes, where they are intentionally incorporated as solvents and fragrance fixatives. Primary phthalates used in cosmetic products include di-butyl phthalate (DBP), which is used as a stabilizer in nail polishes, hair sprays, and perfumes\u003csup\u003e7\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStructurally, phthalate esters are defined by a diester configuration, featuring a benzene dicarboxylic acid backbone conjugated to two ester side chains. These compounds are not chemically bound to the plastic matrix, making it easy for them to leach into the surrounding environment including air, water, food, and soil resulting in widespread human and ecological exposure\u003csup\u003e5\u003c/sup\u003e. Human exposure to phthalates occurs via ingestion, inhalation, dermal absorption, and transplacental transfer during pregnancy\u003csup\u003e8\u003c/sup\u003e. DBP has been detected in consumer items in concentrations ranging from 444.6 to 1671.1 \u0026mu;g/mL\u003csup\u003e9\u003c/sup\u003e. The developmental period is particularly sensitive to EDC exposure, as it is characterized by intense cellular differentiation and hormonal regulation. Numerous studies have demonstrated that phthalates disrupt reproductive processes by mimicking or antagonizing natural hormones, leading to abnormalities such as testicular atrophy, impaired spermatogenesis, and reduced fertility. DEHP has been shown to alter the expression of genes involved in testis development and steroidogenesis, with Sertoli cells being primary targets for phthalate-induced damage. Furthermore, immature animals appear more susceptible to these effects than adults, with gonadal damage following DEHP exposure in young rats\u003csup\u003e10\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn addition to reproductive toxicity, phthalates have increasingly been implicated in sexual, neurobehavioral and psychiatric disturbances. The global rise in neuropsychiatric disorders may be partially explained by the pervasive use of EDCs such as phthalates, bisphenols, and vinclozolin\u003csup\u003e11\u003c/sup\u003e.\u0026nbsp;DBP is a male-specific reproductive toxicant\u003csup\u003e5\u003c/sup\u003e, while DEHP is linked to reduced play in boys, elevated metabolites in autistic children, and anxiety- and depression-like behaviors in animals\u003csup\u003e10\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOne of the most concerning findings about EDCs is their ability to produce intergenerational and transgenerational effects. Phthalates can influence germline epigenetic programming, leading to inherited reproductive and behavioral abnormalities across multiple generations. These include reduced sperm counts, altered stress hormone levels, impaired social behaviors, and disrupted brain development in descendants never directly exposed to the chemical\u003csup\u003e10,12\u003c/sup\u003e. Mechanistically, EDCs act through complex pathways, including genomic and non-genomic nuclear receptor activation, ion channels, nonsteroidal receptors, and transcriptional regulators\u003csup\u003e12\u003c/sup\u003e. These chemicals bind to receptors meant for naturally produced (endogenous) hormones that are essential to everything from growth to reproduction \u003csup\u003e2\u003c/sup\u003e.\u0026nbsp;Given the alarming rise in phthalate consumption such as a 30% increase in phthalic anhydride use over the two past decades\u003csup\u003e13\u003c/sup\u003e and the mounting evidence of their reproductive and behavioral toxicity, further studies are needed to clarify the scope and mechanisms of their effects. Owing to their extensive and prevalent application in a wide range of consumer and industrial products, DEHP and DBP were selected as representative compounds for investigation in the present study, aiming to examine behavioral consequences of prepubertal exposure\u0026shy;\u0026shy;\u0026shy;\u0026shy; in male mice, with a specific focus on the potential for transgenerational transmission of these effects through the male germ line (sperm). By comparing the impacts on both reproductive potential and sexual behavior across generations, this research seeks to contribute to the understanding of how phthalate exposure during development may have lasting implications for and fertility.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cstrong\u003e2.1.\u0026nbsp;Sex hormones level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.1. \u0026nbsp; At 36 days of age\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFSH levels significantly increased in all treatments compared to control, exerting the highest effect by the median dose (200 mg/kg/bw). The lowest (100 mg/kg/bw) and highest (400 mg/kg/bw) doses were lesser effective than the median one, respectively, yet remaining significantly elevated above control levels (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, F\u003csub\u003e6, 14\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e221.5, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.989576; Fig. 2a).\u003c/p\u003e\n\u003cp\u003eLH levels were significantly elevated in DBP-treated groups compared to the control, with the highest observed level in DBP (200 mg/kg/bw), followed by DBP (100) and DBP (400). In contrast, DEHP-treated groups exhibited a moderate dose dependent increase in LH levels. DEHP (400) showed a significant elevation compared to control (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001, F\u003csub\u003e6, 14\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e110.8, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.979375), whereas DEHP (100) and DEHP (200) were insignificantly different from control (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05; Fig. 2b).\u003c/p\u003e\n\u003cp\u003eA significant reduction in serum free testosterone levels was observed in all treated groups compared to the control (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, F\u003csub\u003e6, 14\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e2166, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.998924). Both DBP and DEHP induced a dose-dependent decline, with the highest dose (400 mg/kg/bw) causing the most pronounced decrease, showing insignificant difference among DBP and DEHP (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05; Fig. 2c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.2. \u0026nbsp; At 50 days of age\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFSH levels showed a general decline compared to the elevated levels observed at 36 days, indicating improvement in hormonal regulation. In DBP groups, FSH levels were significantly increased only at the 100 mg/kg/bw dose, while DBP 200 and 400 returned to near control levels. In contrast, DEHP groups exhibited a dose dependent increase in FSH: DEHP 100 showed insignificant change, DEHP 200 showed a significant elevation, and DEHP 400 displayed a highly significant increase. Overall, FSH levels were improved by day 50, particularly in DBP 200 and 400 groups, while higher doses of DEHP continued to elevate FSH, indicating a delayed recovery at high exposure (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, F\u003csub\u003e6, 14\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e37.21, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.940993; Fig. 3a).\u003c/p\u003e\n\u003cp\u003eLH levels showed a general decline compared to the elevated levels observed at 36 days, indicating partial hormonal recovery. However, LH remained significantly elevated in all treated groups compared to control. In DBP groups, LH levels increased in a dose dependent manner. In DEHP groups, a more pronounced dose dependent increase was observed with the highest dose (400 mg/kg/bw) showing the highest LH level. Overall, while LH levels at day 50 were lower than that at day 36, especially in DBP groups, they remained significantly elevated particularly in DEHP 400 suggesting incomplete but notable hormonal improvement over time (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, F\u003csub\u003e6, 14\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e29.42, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.926517;\u0026nbsp;Fig. 3b).\u003c/p\u003e\n\u003cp\u003eFree testosterone levels showed partial recovery compared to the lower levels observed at 36 days. However, levels in all DBP and DEHP groups remained significantly lower than control. Among DBP-treated groups, DBP (100 mg/kg/bw) showed the highest improvement, while DBP 200 remained the lowest. DBP 400 showed moderate recovery. In DEHP treated groups, free testosterone levels remained consistently low, with DEHP 200 showing the least recovery, while DEHP 100 and 400 showed similar levels. Overall, free testosterone showed some improvement by day 50, particularly in DBP 100 and 400 groups, but hormone levels did not return to control values (\u003cem\u003ep\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003e\u0026lt;\u0026nbsp;\u003c/em\u003e0.0001, F\u003csub\u003e6, 14\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e903.6, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.997424; Fig. 3c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.\u0026nbsp;Immunohistochemical expression of estrogen (ER\u003c/strong\u003e\u003cstrong\u003e\u0026alpha;\u003c/strong\u003e\u003cstrong\u003e) and androgen (AR) receptors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1. \u0026nbsp; At 36 days of age\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1.1. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Estrogen receptors (ER\u0026alpha;)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn control testes, ER\u003c/em\u003e\u0026alpha;\u003cem\u003e\u0026nbsp;expression was largely restricted to Leydig cells cytoplasm, with minimal staining in\u0026nbsp;\u003c/em\u003eperitubular myoid layer (PTL)\u003cem\u003e\u0026nbsp;(Fig. 4a). In treated groups, ER\u003c/em\u003e\u0026alpha;\u003cem\u003e\u0026nbsp;expression was markedly increased: DBP 200 showed the most extensive and intense staining across Leydig, Sertoli, spermatogonia and\u0026nbsp;\u003c/em\u003ePTL\u003cem\u003e\u0026nbsp;(Fig. 4c).\u0026nbsp;\u003c/em\u003eAt the highest dose (400 mg/kg) of DBP and DEHP, staining remained strong in Sertoli cells, spermatogonia, and the PTL, \u003cstrong\u003ebut Leydig cells were not detected\u003c/strong\u003e (Fig. 4d \u0026amp; g).\u003cem\u003eThe remaining treated groups displayed ER\u003c/em\u003e\u0026alpha;\u003cem\u003e\u0026nbsp;expression mainly in Leydig cells and\u0026nbsp;\u003c/em\u003ePTL\u003cem\u003e, but with markedly increased intensity compared to control (Fig. 4 b,e\u0026amp;f).\u0026nbsp;\u003c/em\u003eThese qualitative observations were confirmed by quantitative analysis of optical density (OD), which showed a statistically significant increase in ER\u0026alpha; expression in all treated groups compared to control (\u003cem\u003ep=0.0007\u003c/em\u003e, F\u003csub\u003e6, 63\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e12.61, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.545651; Fig. 4h). Importantly, ER\u0026alpha; expression was consistently higher in all DBP treated groups than in the corresponding DEHP treated groups across all doses. Among these, the DBP (200) dose induced the most pronounced upregulation of ER\u0026alpha; expression. Although DEHP treatment at all doses (100, 200, and 400) resulted in significantly higher ER\u0026alpha; expression than the control group, no statistically significant differences were observed among the DEHP doses themselves (\u003cem\u003ep\u0026gt;0.05\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1.2. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Androgen receptors (AR)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn control testes, AR expression was highly pronounced in the nuclei of Sertoli cells, Leydig cells cytoplasm and\u0026nbsp;\u003c/em\u003ePTL\u003cem\u003e\u0026nbsp;(Fig. 5a). In treated groups, AR expression was inhibited in the seminiferous tubules and was primarily confined to Leydig cells and\u0026nbsp;\u003c/em\u003ePTL\u003cem\u003e\u0026nbsp;(Fig. 5 b-e).\u0026nbsp;\u003c/em\u003eHowever, in the DEHP 200 and 400 groups, Leydig cells were absent, with AR staining confined to the PTL\u003cem\u003e\u0026nbsp;(Fig. 5 f\u0026amp;g)\u003c/em\u003e.\u003cem\u003e\u0026nbsp;\u003c/em\u003eStatistically, all treated groups showed a significant decrease in AR expression compared to control group (\u003cem\u003ep\u0026lt; 0.0001\u003c/em\u003e, F\u003csub\u003e6, 63\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e10.12, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=\u0026nbsp;0.490786; Fig. 5h). However, the lower dose of DBP (100) as well as the low and high doses of DEHP (100 and 400) displayed the lowest AR expression among all treated groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2. \u0026nbsp; At 70 days of age\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2.1. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Estrogen receptors (ER\u0026alpha;)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eER\u0026alpha; expression in control testes was confined to Leydig cells cytoplasm, with no detectable staining in the seminiferous tubules\u003cem\u003e\u0026nbsp;(Fig. 6a)\u003c/em\u003e. In treated groups, ER\u0026alpha; expression persisted in Leydig cells cytoplasm and was also observed in some Sertoli cells \u003cem\u003e(Fig. 4 b-g)\u003c/em\u003e, with DBP 200 additionally showing expression in spermatogonia and spermatids \u003cem\u003e(Fig. 6c)\u003c/em\u003e, indicating continued upregulation in these cells. Statistical analysis of OD revealed that ER\u0026alpha; expression was significantly increased only in the DBP 100 group compared with the control (\u003cem\u003ep= 0.0159\u003c/em\u003e, F\u003csub\u003e6, 63\u0026nbsp;\u003c/sub\u003e= 2.856, ƞ\u003csub\u003ep\u003c/sub\u003e\u0026sup2; = 0.214; Fig. 6h). Although other treated groups showed elevated ER\u0026alpha; levels, these increases were statistically insignificant (\u003cem\u003ep\u0026gt;0.05\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2.2. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Androgen receptors (AR)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAR expression in control testes showed no detectable staining within the seminiferous tubules and was confined to Leydig cells cytoplasm (Fig. 7a). In treated testes, abnormal AR expression was observed, with some staining present in Sertoli nuclei (Fig. 7 b,c,e\u0026amp;f); however, the highest dose (400) of both DBP and DEHP did not exhibit any AR expression within the seminiferous tubules, while expression in Leydig cells cytoplasm was maintained (Fig. 7 d\u0026amp;g).\u0026nbsp;\u003c/em\u003eStatistically, analysis of OD revealed a persistent significant decrease in AR expression in most treated groups compared to control (\u003cem\u003ep=0.0038\u003c/em\u003e, F\u003csub\u003e6, 63\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e3.615, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=\u0026nbsp;0.256111; Fig. 7h). The lowest AR expression among treated groups was observed in the highest and lowest doses of DBP (100 and 400) and the intermediate dose of DEHP (200).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.\u0026nbsp;Effects of phthalate exposure on sexual behavior across generations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.1. \u0026nbsp; F\u003c/strong\u003e\u003cstrong\u003e₀\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Generation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.1.1. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Mount latency \u0026amp; frequency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAlthough mount latency tended to increase in both phthalate treated groups relative to controls, particularly in the DEHP group, these differences were not statistically significant\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cem\u003ep=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/em\u003e0.0563, F\u003csub\u003e2, 6\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e4.829, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.616809; Fig. 8a). Mount frequency were significantly affected. Both DBP and DEHP groups exhibited a significant increase in mount frequency compared to control group (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001, F\u003csub\u003e2, 15\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e24.50, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.765625). No significant difference was detected among DBP and DEHP groups (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05; Fig. 8b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.1.2. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Intromission latency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntromission latency was significantly increased in the DBP and DEHP groups compared to control (\u003cem\u003ep=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/em\u003e0.0130, F\u003csub\u003e2, 15\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e5.889, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.439839), indicating a delay in initiating successful copulatory behavior. No significant difference was observed among DBP and DEHP groups (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05; Fig. 8c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.1.3. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Ejaculation latency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEjaculation latency was significantly reduced in DBP and DEHP groups compared to control (\u003cem\u003ep=\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e0.0035, F\u003csub\u003e2, 15\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e8.466, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.530252), with insignificant difference between DBP and DEHP groups (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05; Fig. 8d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.1.4. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Post ejaculatory interval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePost ejaculatory interval was significantly reduced in DEHP group compared to control and DBP groups (\u003cem\u003ep=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e0.001\u003c/em\u003e, F\u003csub\u003e2, 15\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e11.46, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.60443). Insignificant difference was observed among control and DBP groups (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05; Fig. 8e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.1.5. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Inter-mating Interval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInter-mating interval was significantly increased in DBP and DEHP groups compared to control (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). DBP group showed the longest interval, which was significantly longer than both control and DEHP groups. Additionally, DEHP group had a significantly longer interval than control but was significantly shorter than DBP group (\u003cem\u003ep=\u0026nbsp;\u003c/em\u003e0.0401, F\u003csub\u003e2, 6\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e5.763, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.657651; Fig. 8f).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2. \u0026nbsp; F\u003csub\u003e1\u003c/sub\u003e\u003c/strong\u003e \u003cstrong\u003eGeneration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2.1. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Mount Latency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMount latency showed insignificant increase across treated groups compared to control, with more pronounced elevations observed in the TC(B/E) and TT(B) groups\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cem\u003ep=\u0026nbsp;\u003c/em\u003e0.5443, F\u003csub\u003e6, 14\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e0.8637, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=\u0026nbsp;0.270157; Fig. 9a).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2.2. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Mount Frequency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMount frequency was significantly increased in TC (B/E) and TT (B/E) groups compared to control group (\u003cem\u003ep \u0026lt;\u0026nbsp;\u003c/em\u003e0.0001, F\u003csub\u003e6, 35\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e182.3, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.968994). TT (B) group exhibited the highest mount frequency among all other groups. TC (E) group also showed a significantly higher frequency than control, CT (B/E) groups. No significant difference was observed among control and both CT (B/E) groups (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05; Fig. 9b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2.3. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Intromission Latency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntromission latency was significantly increased in TC (B/E) and TT (E) groups compared to control (\u003cem\u003eP\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u0026lt;\u0026nbsp;\u003c/em\u003e0.0001, F\u003csub\u003e6, 35\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e10, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.631579). TT (E) group showed the longest latency among all other groups. In contrast, insignificant differences were observed among control and CT (B/E) and TT (B) groups (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05; Fig. 9c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2.4. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Ejaculation Latency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEjaculation latency was significantly altered across treated groups compared to control. CT (B/E) groups showed significantly longer ejaculation latency than control. In contrast, TC (B/E) and TT (B/E) groups exhibited significantly shorter ejaculation latency than control. Among them, TT (B) being the most reduced (\u003cem\u003ep\u003c/em\u003e\u003cem\u003e\u0026lt; 0.0001\u003c/em\u003e, F\u003csub\u003e6, 35\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e116.1, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.95216; Fig. 9d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2.5. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Post ejaculatory Interval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePost ejaculatory interval was significantly reduced in all treated groups compared to control. TT (B) and TC (E) groups showed the most pronounced reductions, with values significantly lower than other groups (\u003cem\u003ep \u0026lt; 0.0001\u003c/em\u003e, F\u003csub\u003e6, 35\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e52.37, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.899777; Fig. 9e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2.6. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Inter-mating Interval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInsignificant differences were observed among control and all treated groups. Among all groups, TC (E) exhibited the highest inter-mating interval, while other exposed groups showed intermediate changes compared to control (\u003cem\u003ep=\u0026nbsp;\u003c/em\u003e0.6809, F\u003csub\u003e6, 14\u003c/sub\u003e=\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e0.6627, ƞ\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.221192; Fig. 9f).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.3. \u0026nbsp; Sexual motivation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder normal physiological conditions, male mice exhibit mating behavior approximately every two to three days depending on the strain; a pattern consistently observed in the control group. Video recordings confirmed that control males successfully mated with both females and made multiple escape attempts, ultimately succeeding, which reflects normal sexual motivation and power. The escaped male gained access to a separately housed female outside the primary experimental setup, and that female subsequently became pregnant. Control males employed two distinct escape strategies driven by sexual motivation: (1) repeatedly jumping to cling to the wire-mesh ceiling and gnawing the wax seal (videos 1\u0026amp;2), and (2) persistently enlarging the water inlet until it was wide enough to escape (video 3). In contrast, treated males displayed a marked delay in initiating mating behavior, with intervals extending up to 13 days. Notably, some males in TT (B) group failed to initiate mating altogether during the observation period, and exhibited no escape attempts, indicating a potential reduction in sexual drive and behavioral responsiveness.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.4. \u0026nbsp; Circadian rhythm\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA clear disruption of circadian rhythm was observed in most treated groups across both generations, marked by a temporal shift in mating activity from the evening, as seen in controls, to the early morning. In control males, mating activity peaked around 9.00 p.m. (video 4), whereas treated groups exhibited a marked shift, with copulatory behavior occurring predominantly between 4.00 and 7.00 a.m. (videos 5-8). This alteration was accompanied by an inverted sleep\u0026ndash;wake cycle, with both mating and general locomotor activities occurring predominantly during morning rather than during nocturnal phase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.5. \u0026nbsp; Digging \u0026amp; jumping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn both F₀ (parental) and F₁ (offspring) generations, a range of abnormal behaviors were consistently observed in the treated groups, indicating possible \u003cstrong\u003etransgenerational effects\u003c/strong\u003e of phthalate exposure. One of the most frequent behaviors was \u003cstrong\u003eexcessive and repetitive digging\u003c/strong\u003e, particularly in sawdust bedding. This behavior was more pronounced in DEHP treated animals across both generations (videos 9\u0026amp;10). Along with \u003cstrong\u003eabnormal repeated jumping and hyperactivity (\u003c/strong\u003evideos 11\u0026amp;12)\u003cstrong\u003e,\u003c/strong\u003e behaviors that were absent or minimal in control group. These signs of altered activity patterns were consistent across both generations in DBP treated animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.6. \u0026nbsp; Homosexuality\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNotably, female\u0026ndash;female sexual behaviors, such as mounting and mating attempts, were observed exclusively in (TT) groups and were absent in controls. This behavior was particularly pronounced in the F₁ generation (videos 13\u0026amp;14). It is worth noting that the male that did not exhibit any mating attempts was housed with females that displayed female\u0026ndash;female mounting behavior.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.7. \u0026nbsp; Feminization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder normal conditions, when a group of females is housed with a single male, females usually sleep close to each other while the male rests separately (video 15). In the present experiment, however, some DBP treated males were observed sleeping in close contact with females, which may reflect feminization related behavioral alterations (video 16), an atypical behavior which was not noted in control males, which predominantly slept alone. Although this behavior was not frequently observed, its presence represents a deviation from typical male resting patterns.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMoreover, males in the F\u003csub\u003e1\u003c/sub\u003e generation TT (B) displayed no mounting attempts, further indicating the persistence of feminized behavior in offspring derived from DBP exposed males.\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe present study demonstrates that paternal exposure to phthalates in the F₀ generation induced persistent endocrine disruption, characterized by reduced testosterone, compensatory elevations in LH and FSH, ER\u0026alpha; upregulation, and AR attenuation. These hormonal alterations were accompanied by sexual and neurobehavioral impairments, including delayed mating, reduced sexual motivation, feminization, and autism-like behaviors, which persisted into the F₁ generation.\u003c/p\u003e\n\u003cp\u003eThe observed reductions in free testosterone, along with compensatory elevations in LH and FSH, are indicative of potential primary testicular failure (hypogonadism), wherein the testes fail to adequately produce sperm or synthesize testosterone. Consistent with previous studies who established that testosterone, the principal androgen produced by Leydig cells, is essential for spermatogenesis and normal male sexual behavior, and that disruption of androgen signaling can impair copulatory efficiency and sexual motivation \u003csup\u003e14,15\u003c/sup\u003e\u003ca id=\"_anchor_1\" href=\"#_msocom_1\" language=\"JavaScript\" name=\"_msoanchor_1\"\u003e\u003c/a\u003e. Moreover, exposure to phthalate metabolites has been linked to adult-onset hypogonadism and endocrine disruption, affecting gonadotropin balance and testicular function \u003csup\u003e16,17\u003c/sup\u003e. Specifically previous studies\u003csup\u003e18\u003c/sup\u003e have reported phthalate induced suppression of key steroidogenic genes and proteins, suggesting that the observed severe testosterone deficiency in the present study may be a consequence of phthalate induced disruption of the genes responsible for its biosynthesis.\u003c/p\u003e\n\u003cp\u003eTestosterone exerts its biological effects primarily through the androgen receptor, which is expressed in Leydig cells, Sertoli cells, peritubular myoid cells, and germ cells\u003csup\u003e19\u003c/sup\u003e. Disruption of AR function, whether through genetic mutations or receptor loss, results in androgen insensitivity, testicular feminization, and impaired spermatogenesis, as demonstrated in AR knockout models\u003csup\u003e20\u003c/sup\u003e. Reduced AR expression in Sertoli and peritubular myoid cells has also been reported in cases of non-obstructive azoospermia, highlighting the essential role of AR signaling in maintaining spermatogenic integrity\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePhthalates are well established EDCs with pronounced anti-androgenic properties. Experimental studies have shown that phthalates inhibit AR activity, act as antagonists in reporter gene assays, and interfere with receptor dimerization and downstream transcriptional signaling, ultimately suppressing androgen dependent gene expression in Sertoli and germ cells\u003csup\u003e22\u003c/sup\u003e. Also, recent study reporting that elevated urinary DBP metabolite levels are associated with altered male reproductive hormones\u003csup\u003e23\u003c/sup\u003e, supporting the endocrine-disrupting effects of phthalates. The markedly reduced testosterone levels observed in the present study further confirm the suppression of androgenic signaling induced by DBP and DEHP exposure.\u003c/p\u003e\n\u003cp\u003eIn the present study, attenuation or absence of AR expression in treated testes is likely attributable to the anti-androgenic actions of phthalates. Interestingly, while AR expression in control testes at 70 days of age was relatively low, consistent with the attainment of endocrine stability at sexual maturity, the re-emergence of AR expression in a subset of Sertoli cells in treated animals may represent a compensatory response to earlier androgen suppression. Such delayed or atypical receptor re-expression has been interpreted as a cellular attempt to restore diminished androgenic signaling\u003csup\u003e24\u003c/sup\u003e. However, at the highest phthalate dose (400 mg/kg/bw), toxic effects may override these compensatory mechanisms, consistent with established dose dependent toxicity principles\u003csup\u003e25\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eDespite attenuated AR expression, spermatogenesis persisted in treated testes, coinciding with enhanced ER\u0026alpha; expression. Estrogens play a critical role in male reproductive physiology through modulation of the hypothalamic\u0026ndash;pituitary\u0026ndash;gonadal axis and local regulation of spermatogenesis within the testis\u003csup\u003e26\u003c/sup\u003e. Both ER\u0026alpha; and ER\u0026beta; are expressed in Leydig cells, Sertoli cells, peritubular myoid cells, and germ cells, with species-specific distribution patterns\u003csup\u003e27\u003c/sup\u003e. Estrogen signaling through these receptors regulates germ cell differentiation, survival, and apoptosis, thereby coordinating spermatogenic progression \u003csup\u003e27,26\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn the current study, ER\u0026alpha; overexpression was observed in treated testicular sections, particularly in the DBP treated groups. Alterations in ER\u0026alpha; expression have been associated with pathological conditions of the male gonad. Increased ER\u0026alpha; expression in Leydig cells has been reported in cryptorchid testes, suggesting that excessive estrogen signaling contributes to impaired testicular function\u003csup\u003e28\u003c/sup\u003e. Similarly, \u003cem\u003ein utero\u003c/em\u003e DBP exposure has been shown to increase ER\u0026alpha; expression while reducing ER\u0026beta; and AR expression, indicating a shift toward estrogen dominant signaling within the testicular microenvironment\u003csup\u003e29\u003c/sup\u003e. DEHP exposure has also been reported to alter testicular gene expression profiles related to hormonal signaling and receptor regulation\u003csup\u003e30\u003c/sup\u003e. Also, latest evidence indicates that that phthalates exhibited AR antagonist effect based on non-AR dimerization and agonistic effect of ER\u0026alpha; \u003csup\u003e31\u003c/sup\u003e. In agreement with these reports, the present study suggests that the observed estrogen overexpression may be explained by two converging mechanisms: a direct estrogenic action of phthalates and an indirect effect mediated by phthalate induced androgen deficiency, which removes androgen dependent inhibitory control over estrogen signaling. Together, these mechanisms promote ER\u0026alpha; overexpression and estrogen-dominant signaling.\u003c/p\u003e\n\u003cp\u003eIn the present study, phthalate exposure, directly in F₀ and indirectly in F₁ generations, resulted in significant alterations in male sexual behavior, particularly affecting timing and structure of mating activity. Increased mount and intromission latencies indicated a delay in the initiation of effective copulatory behavior, while elevated mount frequencies observed in the treated groups may reflect heightened, yet disorganized or inefficient, sexual attempts. These findings are consistent with earlier studies reported that phthalate exposure induced increased intromission latency and larger number of intromissions to achieve ejaculation\u003csup\u003e32\u003c/sup\u003e, indicating that phthalates adversely affected the efficiency of copulatory behavior.\u003c/p\u003e\n\u003cp\u003eThe significant reduction in ejaculation latency in treated groups, alongside a shortened post ejaculatory interval, suggests an alteration in the refractory phase of sexual behavior, potentially impairing normal sexual pacing. This reduction may reflect a disruption in neuroendocrine regulation of sexual satiety or recovery mechanisms. Several studies have reported that phthalate exposure induced anxiety like behaviors in rodents\u003csup\u003e33\u003c/sup\u003e. Consistent with these findings, behavioral assessments in the present study revealed that treated mice displayed clear indicators of anxiety. Previous experiments have shown that anxiety in male mice is associated with a significant reduction in ejaculation latency\u003csup\u003e34\u003c/sup\u003e. Therefore, it is plausible that the ejaculation latency reduction observed in the present phthalate groups may be mediated by phthalate induced anxiety. In another study, phthalate exposure likely determines epigenetic\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ealterations in programming of sex brain differentiation and regulation of testicular steroidogenesis, leading to long-term reproductive and behavioral disorders\u003csup\u003e35\u003c/sup\u003e. \u0026nbsp;\u003cstrong\u003eIn agreement with\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethose studies, it is suggested that phthalate-induced anxiety, neuroendocrine disruption, and epigenetic reprogramming likely act together to shorten ejaculation latency and impair sexual pacing.\u003c/p\u003e\n\u003cp\u003eThe present observations revealed a prolonged inter-mating interval reaching delays of up to 13 days in certain treated groups TC (E). Control male mice typically resume mating within hours to a few days depending on the strain; for instance, C57BL/6 males average approximately 4 days, while DBA/2 males may remate within an hour\u003csup\u003e36\u003c/sup\u003e. A prolonged interval of this magnitude therefore signals a marked disruption in reproductive function, likely reflecting a combination of reduced sexual motivation, neurobehavioral impairment, and physiological dysfunction. Such alterations are consistent with previous findings that EDCs such as DBP and DEHP interfere with androgen signaling, suppress testosterone synthesis, and disrupt central neuroendocrine circuits regulating sexual behavior\u003csup\u003e37\u003c/sup\u003e. Recently, it was found that many male rats exposed to phthalates \u003cem\u003ein utero\u003c/em\u003e and during lactation were sexually inactive in the presence of receptive females, and this inactivity was not associated with morphological abnormalities, suggesting a disturbance in sexual brain differentiation\u003csup\u003e35\u003c/sup\u003e. Notably, insignificant differences were found between DBP and DEHP groups for most parameters, suggesting that both phthalates exert comparable disruptive effects on timing and coordination of sexual performance.\u003c/p\u003e\n\u003cp\u003eSexual motivation in rodents is a well-established model for studying reward-driven behavior. The escape behavior observed in the current control male mice, leaving the cage to access a receptive female indicates strong sexual motivation rather than exploratory activity. Operant paradigms show that males will repeatedly nose-poke or lever-press to gain access to a mate, reflecting high effort expenditure for sexual reward\u003csup\u003e38\u003c/sup\u003e. Similarly, the electrified grid barrier model, first described in early behavioral studies\u003csup\u003e39\u003c/sup\u003e, demonstrated that males would endure aversive electrical stimulation to approach a female,\u0026nbsp;underscoring that sexual motivation can override discomfort an observation consistent with current findings. In contrast, this escape response was absent in all phthalate males, providing strong evidence that phthalate exposure reduces sexual motivation.\u0026nbsp;Reduced androgen receptor expression and testosterone levels likely underlie this loss of sexual motivation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn the present study, female\u0026ndash;female sexual behaviors were observed exclusively in the F\u003c/strong\u003e\u003cstrong\u003e₁\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;generation.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eA similar phenomenon has been reported, with the emergence of homosexual-type behaviors in male rats prenatally exposed to low doses of DBP\u003csup\u003e35\u003c/sup\u003e. In their study, 18-month-old males exhibited testicular and accessory gland atrophy, reduced testosterone, increased Leydig cell adenomas, and impaired sexual performance. These effects were linked to prenatal DBP-induced epigenetic changes disrupting brain sexual differentiation and testicular steroidogenesis. By analogy, the female\u0026ndash;female sexual interactions documented in the current research may similarly reflect phthalate-induced alterations in neuroendocrine pathways governing sexual behavior. Such disruptions could originate from changes in the organizational effects of sex steroids during critical developmental periods, ultimately leading to atypical sexual behaviors and potential reproductive dysfunction later in life.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEstrogen signaling plays a key role in shaping social affiliation and sexual behavior, and excessive ER\u0026alpha; activation during critical developmental windows has been shown to bias neural circuits toward feminized behavioral outputs\u003csup\u003e40,27\u003c/sup\u003e.\u003cem\u003e\u0026nbsp;\u003c/em\u003eIn the present study, treated males exhibited increased affiliative proximity to females during rest, a clearly feminized social behavior alongside ER\u0026alpha; expression in the testicular tissue, \u003cem\u003esuggests a relative estrogenic shift resulting from altered estrogen\u0026ndash;androgen signaling. Disruption of this hormonal balance is known to interfere with male sexual differentiation and behavioral organization, and altered estrogen signaling has been associated with changes in male typical behaviors following prenatal phthalate exposure in humans\u003csup\u003e40\u003c/sup\u003e. Also, phthalate exposure has been linked to feminization related behavioral alterations and increased anxiety like behavior\u003csup\u003e33\u003c/sup\u003e.\u003c/em\u003e Notably, in the present study, DBP exposure induced a more pronounced ER\u0026alpha; expression than DEHP, supporting previous evidence that DBP preferentially enhances estrogen receptor mediated signaling while suppressing androgenic pathways\u003csup\u003e29,31\u003c/sup\u003e. This molecular difference was reflected behaviorally, as DBP treated males exhibited more overt feminized behaviors, whereas such behaviors were less evident in DEHP exposed groups. \u003cem\u003eConsistent with this interpretation\u003c/em\u003e, endocrine signaling is compelled to shift toward one of two hormonal pathways. Consequently as discussed above, the loss of androgenic signaling leaves estrogen signaling as the dominant and functionally main pathway, promoting ER\u0026alpha; overexpression and estrogen dominant signaling, which may underlie the observed feminized behavior in male mice.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In contrast, DEHP exposure was more closely associated with neurobehavioral abnormalities, including repetitive and anxiety like behaviors\u003csup\u003e41,42,1\u003c/sup\u003e, suggesting preferential disruption of neurodevelopmental pathways.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eImportantly, the persistence of altered sexual and social behaviors in both the F₀ and F₁ generations may be attributed to disruption of the pubertal organizational period, which has been described as a second critical window during which gonadal hormones permanently modify neural processing in brain regions regulating sexual behavior\u003csup\u003e14\u003c/sup\u003e. Collectively, these findings indicate that phthalate exposure induces estrogen receptor overexpression alongside attenuation of androgenic signaling in the male gonad, thereby contributing to disrupted sexual differentiation and feminized behavioral traits. This highlights the critical importance of balanced estrogen\u0026ndash;androgen signaling in maintaining normal male reproductive and behavioral phenotypes and underscores the potential long-term consequences of phthalate exposure on male endocrine and neurobehavioral health. Overall, these findings highlight a serious concern, suggesting that phthalate exposure could contribute to the emergence of effeminized male traits in human and may represent one of several environmental factors potentially linked to the increasing incidence of homosexuality in certain communities.\u003c/p\u003e\n\u003cp\u003eDBP was first demonstrated to interfere with the normal functioning of circadian rhythm circuits which regulate rhythmic expression of numerous genes\u003csup\u003e17,43\u003c/sup\u003e. Disruption of circadian gene expression has been implicated in the pathogenesis of various diseases, including cancer\u003csup\u003e44\u003c/sup\u003e. These findings are consistent with the current study, in which disruption of biological clock regulation was observed in DBP exposed groups manifested as altered rest-activity cycles and abnormal sleep-wake behaviors, thereby suggesting that DBP induced disruption of circadian gene expression and may contribute to broader physiological and behavioral disturbances. Collectively, sexual behavior impairments, circadian rhythm disturbances, abnormal jumping and feminization were predominantly observed in animals directly exposed to DBP or derived from DBP treated parents, suggesting a compound-specific disruption of reproductive neuroendocrine pathways. In contrast, neurobehavioral abnormalities including repetitive digging were primarily associated with DEHP exposure or parental exposure to DEHP, implicating distinct epigenetic or neurodevelopmental mechanisms in mediating these effects. Recent studies emphasize that phthalate effects are strongly influenced by dose, timing of exposure, and sex, supporting evidence that these chemicals can drive multigenerational and transgenerational alterations in development and reproduction\u003csup\u003e12\u003c/sup\u003e. These findings are relevant for understanding how even low dose exposures to environmental chemicals can affect not just the direct offspring but also future generations.\u003c/p\u003e\n\u003cp\u003eIt is important to consider the effects of environmental exposures on neurodevelopment, as numerous studies have demonstrated their significant impact on autism spectrum disorder (ASD) related behaviors. ASD is characterized by repetitive behaviors, social deficits, communication difficulties, stereotyped patterns of behavior, and heightened anxiety\u003csup\u003e45\u003c/sup\u003e. Prenatal phthalate exposure can disrupt synaptic function, impair learning and memory, and alter circadian rhythms, ultimately increasing the risk of ASD development in children\u003csup\u003e46\u003c/sup\u003e. Similarly, the concept of \u0026ldquo;environmentally vulnerable physiology,\u0026rdquo; emphasizing how environmental factors can exacerbate neurodevelopmental vulnerabilities, has been highlighted\u003csup\u003e47\u003c/sup\u003e. Previous findings using various DEHP exposure models have demonstrated alterations in anxiety-like and social interaction behaviors, particularly in males\u003csup\u003e42\u003c/sup\u003e. Recent evidence suggests that phthalate metabolites might interfere with tyrosine and tryptophan metabolism and consequently affect neurobehavioral development\u003csup\u003e1\u003c/sup\u003e. \u0026nbsp;Male mice exposed to 200 mg/kg DEHP displayed elevated digging behavior and decreased self-grooming, both nonsocial behavioral alterations frequently associated with autism-like phenotypes. Consistent with these results, DEHP exposure during gestational and early postnatal development has been linked to increased anxiety in pubertal male offspring, as evidenced by more time spent in the closed arms of the elevated plus maze\u003csup\u003e33\u003c/sup\u003e. Mechanistically, DEHP regulates gene expression and induces neuronal degeneration in the hippocampus, a brain region critical for learning and memory\u003csup\u003e41\u003c/sup\u003e, which may explain the emergence of repetitive digging or jumping behaviors observed in DEHP treated mice in the present study. Collectively, these findings of repetitive behaviors, impaired locomotion, disrupted circadian rhythms, reduced sexual activity, heightened anxiety, and potential cognitive deficits in phthalate-exposed males parallel ASD-like phenotypes in humans. These results suggest that early-life phthalate exposure disrupts typical neurobehavioral trajectories in a sex-specific manner and may contribute to autism risk in children, either through direct exposure during infancy (e.g., pacifiers and feeding bottles) or through potential transgenerational effects associated with parental plastic use.\u003c/p\u003e\n\u003cp\u003eImportantly, transgenerational impacts were evident in F₂ generation. This observation aligns with the proposed concept that when both parents carry the same environmentally or epigenetically influenced trait, the likelihood of its expression in offspring is significantly increased\u003csup\u003e48\u003c/sup\u003e. Consistent with this principle, the present study found that TT group derived from both a treated male and a treated female displayed the most pronounced alterations across behavioral and physiological parameters, supporting the hypothesis of cumulative or synergistic parental effects mediated through heritable epigenetic modifications. The TC group, in which only the male was treated, showed intermediate effects, while the CT group, where the male was unexposed, exhibited the least abnormalities. These patterns highlight the critical role of parental exposure, particularly male exposure in shaping offspring outcomes and further suggest that phthalate-induced disruptions can be amplified when both parents are affected.\u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cp\u003eThe current study was achieved in accordance with the Egyptian laws and University guidelines for animal care. The National Ethical Committee of Assiut University, Faculty of Science Research Ethics Committee (FSREC), Egypt, has approved all the procedures in the present work with the approval number: 01-2025-0017. All procedures involving animals were conducted in accordance with the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.1.\u0026nbsp;Chemicals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDEHP and DBP, with 98% purity, were purchased from SRL (India). The chemicals were merged into corn oil (used as a vehicle) before administration and were stored at room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.\u0026nbsp;Experimental Animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 81 albino mice (Swiss strain) designated as the F₀ generation were obtained from the animal house of the Faculty of Science, Al-Azhar University, Assiut. The experiment was initiated with the 63 immature males aged 21 days, weighing 8\u0026ndash;12 g on average, which were randomly assigned to seven groups: a control group, the remaining six groups were subdivided into three groups for each of DEHP and DBP. These groups were orally administered, once daily, a volume of 100 \u0026micro;l of corn oil carrying the three doses (100, 200 \u0026amp; 400 mg/kg bw) for fifteen consecutive days. Three mice from each group were euthanized at ages of 36, 50 \u0026amp; 70 days by cervical dislocation without prior anesthesia, in accordance with institutional and international guidelines for the care and use of laboratory animals. At ages of 36 and 50 days, blood samples were collected to measure follicle-stimulating hormone (FSH), luteinizing hormone (LH) and free testosterone (FT). At ages of 36 and 70 days, estrogen receptor alpha (ER\u0026alpha;) and androgen receptor (AR) expression was assessed by immunohistochemical investigation. \u0026nbsp;These doses for both DBP \u0026amp; DEHP were chosen based on previous studies \u003csup\u003e49,50\u003c/sup\u003e respectively. Animals were provided food and water \u003cem\u003ead libitum\u003c/em\u003e under standard laboratory conditions, maintained at a temperature of 23 \u0026plusmn; 2 \u0026deg;C and a 12-hour light/dark cycle. To comply with research ethics, the number of animals used was minimized, and stress on the mice was also reduced by avoiding unnecessary handling and by ensuring that only a single observer was present in the room throughout the experiment. Experiments were carried out in strict compliance with the ethics prepared by INSA and (WHO/UNESCO). \u0026nbsp;Following a 15-day recovery period after treatment cessation and upon reaching sexual maturity, mating commenced.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3.\u0026nbsp;Hormonal measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood was collected from the retro-orbital sinus, centrifuged at 5000 rpm for 20 min; serum was separated and stored at \u0026ndash;20 \u0026deg;C until analysis. Serum concentrations of free testosterone, FSH, and LH were measured using immunoassay kits (Free Testosterone, FSH, and LH; Roche Diagnostics, Switzerland) on a Cobas e411\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eimmunoassay analyzer, following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4.\u0026nbsp;Immunohistochemical investigation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe immunohistochemical investigation was carried out on formalin-fixed testis collected at ages of 36 and 70 days. The specimens were processed, embedded in paraffin, and the tissue sections were cut at 4\u0026mu;m. The sections were treated with 10 mL MoL Tris buffer and 1 mL MoL ethylene-diamine tetra-acetic acid, pH 9.0 for 20 min at 90 \u0026deg;C. The block of endogenous peroxidase was done by incubation the sections with 3% H2O2, followed by preincubation overnight at 4 \u0026deg;C in 1% bovine serum albumin in PBS. The sections were stained for 30 min at 37 \u0026deg;C, using the following antibodies that showed reactivity in mice species: a rabbit polyclonal anti-AR (1:200; ABclonal Technology, Wuhan, China; Cat. No. A16200), and a rabbit polyclonal anti-ER\u0026alpha; (1:200, Affinity Biosciences, Wuhan, China; Cat. No. AF6058), according to the method described previously\u003csup\u003e51\u003c/sup\u003e. Sections were counterstained with haematoxylin, and analyzed using an Honor 400 mobile camera. In parallel, tissue specimens, in whom the primary antibodies were omitted and replaced with buffer, served as negative controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5.\u0026nbsp;Breeding the F0, F1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe F₀ generation comprised three experimental groups: control, DBP, and DEHP. In each group, three immature males were randomly selected from animals exposed to a dose of 100 mg/kg DBP or DEHP, and from the corresponding control group. To produce the F₁ generation, each male was mated with two untreated virgin females weighing 21\u0026ndash;25 g, resulting in six females per group (18 in total). Thus, the parental generation (F₀) comprised 27 animals. For the F\u003csub\u003e2\u003c/sub\u003e generation, three mating schemes were designed for each treatment group (DBP \u0026amp; DEHP). Control male \u003cstrong\u003e\u0026times;\u003c/strong\u003e indirect treated female CT (B/E), indirect treated male \u003cstrong\u003e\u0026times;\u003c/strong\u003e control female TC (B/E), and indirect treated male \u003cstrong\u003e\u0026times;\u003c/strong\u003e indirect treated female TT (B/E), where (B) indicates DBP treated and (E) indicates DEHP treated parents. In this notation, the first letter always refers to the male and the second to the female (Fig. 1). Each scheme was replicated three times, yielding nine pairs per group in addition to three replicates of the control \u003cstrong\u003e\u0026times;\u003c/strong\u003e control group. In total, 21 mating pairs (42 animals) were used to generate the F₂ offspring. Mating of the F\u003csub\u003e0\u003c/sub\u003e generation was conducted during the period from (22-4-2024) to (12-5-2024) in the DBP lineage and from (12-5-2024) to (31-5-2024) in the DEHP lineage. Likewise, mating of the F\u003csub\u003e1\u003c/sub\u003e generation occurred from (5-7-2024) to (24-7-2024) in the DBP lineage and from (27-7-2024) to (15-8-2024) in the DEHP lineage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.6.\u0026nbsp;For behavioral assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach male was individually housed in a glass cage with wire mesh for ventilation under controlled light/dark conditions. A video camera was activated for one hour, after which two females were introduced into the cage and cohabitated with the male for 20 days. Body weights of both males and females were recorded daily during this period. Sexual behavior was evaluated by measuring parameters including mount latency \u0026amp; frequency, intromission latency, ejaculation latency, post-ejaculatory interval and Inter-mating Interval according to\u003csup\u003e52\u003c/sup\u003e. Any abnormal or atypical behavior was also noted. Behavioral observations were recorded using a HiLook 2 MP surveillance camera (resolution. 1920\u0026times;1080 pixels, frame rate. 30 fps), at the Zoology \u0026amp; Entomology Department, Faculty of Science, Assiut University.\u003c/p\u003e\n\u003cp\u003eAll experimental procedures applied to the F₀\u0026nbsp;generation\u0026mdash;including video recording, and behavioral assessments\u0026mdash;were consistently replicated for the F₁\u0026nbsp;generation. All procedures involving animals were conducted in accordance with the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.7.\u0026nbsp;Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were estimated as mean \u0026plusmn; SE. Hormonal data and optical density (OD) were analyzed statistically using column statistics and one-way analysis of variance (one-way ANOVA), and the test of Newman-Keuls multiple comparison test as a post test. For sexual behavior assessments, to exclude potential female related effects, a paired t-test was applied to compare measurements obtained from the first and second females mated with the same male. No significant differences were detected between females for any of the assessed parameters. Accordingly, data from both females were considered as biological replicates representing the same male, and group comparisons were subsequently performed using one-way analysis of variance (one-way ANOVA). These analyses were carried out using Prism software for windows, version 5.0 (Graph pad software Inc., San Diego, California, USA) and Excel (Microsoft office 10). Analysis of OD was carried out using the software Image J (the JAVA SE Runtime Enviroment, version 6).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReda A. A. suggested the point, read the manuscript, contributed in interpretation of the results,\u0026nbsp;and\u0026nbsp;revised the manuscript. Dalia Elzahraa F. M. contributed to reviewing the paper. Heba E. A.\u0026nbsp;achieved the practical part\u0026nbsp;and contributed to preparing, writing and reviewing the paper.\u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID for authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReda. A. Ali. ORCID https://orcid.org/0000-0002-6716-9789\u003c/p\u003e\n\u003cp\u003eTelephone No. 00201011423969\u003c/p\u003e\n\u003cp\u003eDalia Elzahraa F. Mostafa ORCID https://orcid.org/0000-0003-0624-9141\u003c/p\u003e\n\u003cp\u003eHeba E. Aboulqasem ORCID https://orcid.org/0009-0002-9199-0662\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHoffman, S. S., Tang, Z., Dunlop, A., et al. Impact of prenatal phthalate exposure on newborn metabolome and infant neurodevelopment. \u003cem\u003eNat Commun.\u003c/em\u003e 16, 2539. https://doi.org/10.1038/s41467-025-57273-z (2025).\u003c/li\u003e\n\u003cli\u003eBrander, S. M. Rethinking our chemical legacy and reclaiming our planet. \u003cem\u003eOne Earth.\u003c/em\u003e 5, 8\u0026ndash;10. https://doi.org/10.1016/j.oneear.2022.03.020 (2022).\u003c/li\u003e\n\u003cli\u003eVan Wezel, A. P., Van Vlaardingen, P., Posthumus, R., Crommentuijn, G. H., Sijm, D. T. H. M. 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Phthalates, including di-butyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP), are widely used industrial chemicals that disrupt hormonal regulation and impair male fertility. This study examined the effects of paternal prepubertal exposure to DBP and DEHP on endocrine function, receptor expression, and sexual behavior in Swiss albino mice. Sixty-three F₀ males (21 days old) were divided into control and treated groups receiving DBP \u0026 DEHP at 100, 200 \u0026 400 mg/kg body weight for 15 days, followed by recovery. Hormonal assays for testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were performed, and estrogen receptor alpha (ERα) and androgen receptor (AR) expression were evaluated immunohistochemically. Low dose exposed males were bred to generate F₁ and subsequently F₂ offspring. Paternal phthalate exposure reduced testosterone, increased LH and FSH, upregulated ERα, and attenuated AR expression, with alterations persisting into adulthood. Behavioral assessments revealed impaired sexual performance, delayed mating, reduced motivation, feminization, homosexuality, autism like traits, and circadian rhythm disruption in F₀ males, effects persisting in F₁ offspring. These findings demonstrate that paternal phthalate exposure induces lasting endocrine, receptor, and behavioral disturbances with multigenerational consequences, underscoring the need for safety evaluation.","manuscriptTitle":"Silent Legacy I: Paternal Phthalate Exposure Shifts Androgen–Estrogen Receptor Balance and Induces Transgenerational Reproductive Disruption, Signs of Autism, Feminization and Homosexuality in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 05:46:40","doi":"10.21203/rs.3.rs-8600132/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"46903911-07d1-451e-a881-e4df03e02cfc","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61395321,"name":"Health sciences/Endocrinology"},{"id":61395322,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-01-30T06:11:01+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 05:46:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8600132","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8600132","identity":"rs-8600132","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}