Chronic Testosterone Administration During Pregnancy Causes Autistic-like Traits in Rat Offspring: Possible Relationships with Cerebral Oxytocin, Dopamine, Serotonin and IGF-1 Levels.

Chronic Testosterone Administration During Pregnancy Causes Autistic-like Traits in Rat Offspring: Possible Relationships with Cerebral Oxytocin, Dopamine, Serotonin and IGF-1 Levels. | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chronic Testosterone Administration During Pregnancy Causes Autistic-like Traits in Rat Offspring: Possible Relationships with Cerebral Oxytocin, Dopamine, Serotonin and IGF-1 Levels. Evrim Senkal, Umut Eryigit, Mehmet Fatih Bozkurt, Volkan Solmaz, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-182999/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 Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects boys three times more frequently than girls. Previous studies have suggested that higher fetal testosterone exposure can cause autism-like behaviors in males. The mechanism of effect of fetal testosterone on autism development is not well known. In the present study, we investigated the relationship between prenatal testosterone exposure and ASD in an experimental study with the use of behavioral tests, histopathological examinations and biochemical measurements performed to compare offspring with and without exposure to testosterone. Female rats were randomly distributed into Group 1 (control, n = 6) and Group 2 (Testosterone undecanoate, n = 6). Female rats were caged with a fertile male (three female/one male) for 2–3 days during the oestrus period. Group 1 rats were given 1 ml/kg % 0.9 NaCl saline on the 10th day of pregnancy, while Group 2 rats were given 250 mg/kg testosterone undecanoate. The dams were allowed to raise their litters until weaning on postnatal day 21 (P21). On P21 , forty littermates (10 male control, ten female control, ten male Testosterone-exposed, and ten female Testosterone-exposed) were randomly separated and housed. These animals underwent behavioral testing in adulthood on P50. Subsequently, the rats were sacrificed. Biochemical analyses [Brain tissue 5-Hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), Insulin-like growth factor-1 (IGF-1), and oxytocin] and histopathological analyses were performed. Analysis of the behavioral tests (three-chamber social test, open field test) revealed significant differences among the groups, with the testosterone-exposed groups demonstrating autistic traits at a greater degree. Histologically, hippocampal CA1 and CA3 regions displayed significant alterations such as gliosis and neuronal cell death in the testosterone-exposed groups compared to controls. Brain levels of tissue 5-HIAA, HVA, IGF-1 increased while oxytocin level decreased in the testosterone-exposed groups. These results suggest a possible link between chronic prenatal testosterone exposure and neurodevelopmental disorders such as ASD. Testosterone exposure and autism-like traits can be linked to dopamine, serotonin, IGF-1 increase, and oxytocin decrease. Endocrinology & Metabolism Neurology Scientific Communication Developmental Neuroscience Autism spectrum disorder (ASD) neurodevelopmental disorder higher fetal testosterone exposure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects communication and behavior. It is characterized by early-onset deficits in social and communication skills, restricted interests and repetitive body movements (Posar et al., 2015). Autism prevalence has been increasing in recent years (Maenner et al., 2020). The etiology of ASD is unknown, but it is thought that genetic and environmental factors come together in the development of ASD (Taylor et al., 2020). Current data demonstrates that boys are affected around three times more frequently than girls (Becker, 2012; Ferri et al., 2018). However, the mechanisms underlying male vulnerability are not well-known and remain understudied. A higher incidence of ASD in males can in fact be among the features that could help to enlighten its etiology. For instance, intrauterine fetal testosterone exposure has gained some recognition as arguably the most popular hypothesis concerning the etiology of autism. Previous studies have suggested that higher fetal testosterone exposure can cause autism-like behaviors in males (Ferri et al., 2018). However, the mechanism between prenatal high dose testosterone exposure and autistic traits is not well documented. Recent studies showed that testosterone induces molecular changes in dopamine in the nigrostriatal pathway, thus increasing the effect of dopamine in the body (Purves-Tyson et al., 2014). It is well known that increased dopamine causes behavioral anomalies (Klein et al., 2019). Serotonin is another neurotransmitter that is shown to increase in autistic children (Anderson et al., 1987). Dysregulation of serotonin has been linked to behavioral anomalies (Hassan et al., 2019), and the latest studies have shown that there is a possible relationship between increased intrauterine testosterone levels and serotonin levels. Oxytocin is a famous molecule that appears to be involved in the neurobiology of autism development as per the results of recent studies. Oxytocin has been shown to modify synaptic plasticity and modulate social behaviors (Insel, 2010). In addition, empathy induction among individuals has been associated with increased oxytocin levels (Procyshyn et al., 2020). IGF-1 (Insulin-like growth factor-1), which reduces neuroinflammation by affecting cytokines and glial activation, is another hormone that can be increased in autism (Riikonen, 2017). This experimental study exposed pregnant rats to testosterone and assessed the results on the offspring via behavioral parameters, such as locomotor activity and anxiety-like behavior, using the open field test and social interaction using the three-chamber test. Additionally, we aimed to evaluate the possible association between prenatal testosterone exposure and other biochemical parameters, including 5-Hydroxyindoleacetic acid (5-HIAA) (serotonin's primary metabolite), homovanillic acid (HVA) (associated with dopamine levels in the brain), oxytocin, and IGF-1. We also evaluated the effect of prenatal testosterone exposure on the cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3) regions of the hippocampus via neuronal morphology, total neuron count, and GFAP immunoexpression. Materials And Methods Animals The experimental procedures employed in the present study were approved by the Animal Ethics Committee (199545685/2). The rats used in the experiment were obtained from the Experimental Animal Laboratory of Bilim University. All experiments were carried out with respect to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (US) (2011). Twelve female and four male Wistar adult rats (220 ± 10 g) were included in the study. The rats were housed in plastic cages and maintained under standard conditions with 12-hour light/dark cycles at room temperature (22 ± 2°C) and a humidity of 45–60%. Study design Female rats were randomly distributed into Group 1 (control, n = 6) and Group 2 (recipents of testosterone undecanoate, n = 6). All animals were monitored daily for behavior and health conditions throughout the study. Female rats were caged with a fertile male (three female/one male) for 2–3 days during the oestrus period. Mating was checked by looking at the white vaginal plaque in rats. Then, male rats were removed from the cages. Group 1 rats were given 1 ml/kg % 0.9 NaCl saline on the 10th day of pregnancy. Group 2 rats were given 250 mg/kg testosterone undecanoate (Nebido Ampul, Bayer, 250 mg/ml) on the 10th day of pregnancy. On the day of birth, to ensure standardized maternal care, litter size was reduced to 9 pups per dam. The dams were allowed to raise their own litters until weaning on postnatal day 21 (P21). On P21, forty littermates (10 male control, ten female control, ten male Testosterone-exposed, and ten female Testosterone-exposed) were randomly separated and housed in same-sex and same study group cages with ad libitum access to standard rodent chow and tap water. These animals underwent behavioral testing in adulthood on P50. All behavioral experiments were conducted between 10.00 AM and 3.00 PM. A summary of the study design is presented in Fig. 1. Behavioral tests Three- chamber sociability and social novelty test The sociability test was performed as previously described with minor modifications (Ellegood and Crawley, 2015; Erbas et al., 2018; Moy et al., 2004). Briefly, a Plexiglas cage (40 cm x 90 cm x 40 cm) was divided into three equal regions (40 cm x 30 cm x 40 cm). On the first day, rats were allowed to habituate in the test cage for 5 min (pre-test session). Twenty-four hours later, to test sociability, a stranger rat was placed inside a small plastic cage with mesh-like holes in one side chamber and an empty cage in the third chamber. Then, the test rat was placed in the center chamber, and the time spent in each region by the test rat was recorded for 10 min (sociability test). The test rat was considered to be in the chamber when its head and two front paws entered the chamber. The field floor was then cleaned between each test with 70% alcohol and dried with a paper towel to remove any traces of olfactory stimuli from the previous rat. Open field test The open-field test was conducted in an open-aired box with dimensions of 50 cm x 50 cm x 40 cm (Erbas et al., 2018). At the beginning of the test, rats were gently placed in the center of the box and freely explored the arena for 5 minutes. Then, each rat was observed for 5 min to evaluate spontaneous activity level. The total number of ambulation (number of floor divisions crossed with the four paws) were recorded. The field floor was then cleaned between each animal with a 70% alcohol-water solution and dried with a paper towel to remove olfactory cues. Hippocampus histopathology The CA1 and CA3 regions of the hippocampus were chosen as the target areas to be examined for hippocampus evaluation. Briefly, following behavioral tests, animals were euthanized, and their brains were removed and fixed in 10% formaldehyde (0.1 M phosphate buffer saline, PBS) for three days. Then, they were moved into 30% sucrose and stored at 4°C until infiltration was complete. The brains were cut coronally on a sliding microtome at 40 µm and mounted on gelatinized glass slides. Cresyl violet staining to quantify the number of surviving neurons were performed in six sections per studied group by an image analysis system (Image-Pro Express 1.4.5, Media Cybernetics, Inc. USA). For GFAP immunohistochemistry, brain sections were incubated with H 2 O 2 (10%) for 30 min to eliminate endogenous peroxidase activity and blocked with 10% normal goat serum (Invitrogen) for one hour at room temperature. Subsequently, sections were incubated in primary antibodies against GFAP (Abcam, Inc., MA, US; 1/1000) for 24 h at 4°C. Antibody detection was performed with the Histostain-Plus Bulk kit (Invitrogen) against rabbit IgG, and 3,3′ diaminobenzidine (DAB) was used to visualize the final product. All sections were washed in PBS and photographed with an Olympus C-5050 digital camera mounted on the Olympus BX51 microscope. To calculate the GFAP immunostaining index, GFAP-positive cells were counted at 40X magnification in randomized Sect. (3–4) for each rat. All histopathological examinations were performed by the same investigator, who was blinded to the study groups. Tissue biochemical analysis After decapitation, brains were rapidly removed and stored at -20°C until biochemical analyses. For tissue analysis, whole cerebral tissues were homogenized with a glass homogenizer in 5X volume of phosphate-buffered saline (pH, 7.4) and centrifuged at 5000 g for 15 min. The supernatant was then collected, and total protein concentration in the brain homogenates was determined according to Bradford ' s method using bovine serum albumin as standard (Bradford, 1976). The brain levels of IGF-1 (Shanghai LZ Biotech Co., Ltd.), oxytocin (Shanghai LZ Biotech Co., Ltd.), 5-HIAA (Shanghai LZ Biotech Co., Ltd.), HVA (Cusabio) in the tissue supernatants were measured using commercially available rat enzyme-linked immunosorbent assay (ELISA) kits. All samples from each animal were measured in duplicate according to the manufacturer ' s guidelines. Statistical analysis Statistical evaluation was performed using SPSS version 15.0 for Windows (SPSS Inc., Chicago, IL, USA). Shapiro-Wilk's W and Levene's tests were used to check normality of distribution and homogeneity of variance, respectively. The results are presented as the mean ± standard error of the mean (SEM). Resultant p-values of < 0.05 were accepted to demonstrate statistical significance. Results Three-Chamber and Open Field Test In the three-chamber sociability and social novelty test, both male and female testosterone-exposed rats spent less time with the stranger rat than the male and female rats of the control group, indicating somewhat less social affiliation and more autism-like phenotype (p < 0.05 for males; p < 0.001 for females). Both male and female testosterone-exposed rats spent significantly less time in the center of the open field area, which suggests increased anxiety and less locomotor activity (p < 0.05 for males ; p < 0.05 for females). Results are shown in Fig. 2. Tissue Biochemical Analysis The concentration of IGF-I, 5-HIAA and HVA measured from brain tissues were significantly higher in both male (p < 0.001; p < 0.05; p < 0.001, respectively) and female (p < 0.001; p < 0.001; p < 0.05, respectively) testosterone-exposed rats compared to control male and female rats. The concentration of oxytocin measured from brain tissues was significantly lower in both male (p < 0.001) and female (p < 0.05) testosterone-exposed rats compared to the male and female rats of the control group (Table-1). Table 1 iochemical results of groups Control group male rats Control group female rats Testosterone exposed male rats Testosterone exposed female rats IGF-1 (U/mg protein) 7.2 ± 0.3 6.3 ± 0.6 24.8 ± 5.5 ** 19.5 ± 3.8 ** Oxytocin (pg/mg protein) 65.1 ± 8.8 70.9 ± 7.4 22.6 ± 7.2** 48.9 ± 10.6 * 5-HIAA (µmol/mg protein) 13.2 ± 2.3 8.8 ± 1.6 33.1 ± 6.7 * 24.5 ± 6.2 ** HVA (ng/mg protein) 0.73 ± 0.09 0.68 ± 0.04 1.2 ± 0.1 ** 0.9 ± 0.3 * Results were presented as mean ± SEM. Statistical analyses were performed by one-way ANOVA. * p < 0.05, ** p < 0.001 different from control groups. Abbreviations: 5-HIAA:5-Hydroxyindoleacetic acid; HVA : Homovanillic acid; IGF-1: Insulin-like growth factor-1; SEM: standard error of the mean. Histopathological evaluation of the groups Decreased neuronal count, and dysmorphological changes in both the CA1 and CA3 regions of the hippocampus, which indicates neuronal body degeneration, were present in both testosterone-exposed male (p < 0.001) and female (p < 0.001) rats. In contrast, the cresyl violet staining results of control group rats demonstrated normal characteristics (Figure-3 for males and figure-4 for females). In both the CA1 and CA3 regions of the hippocampus, GFAP immunoreactivity of hippocampal sections showed increased glial activity in testosterone-exposed male (p < 0.05) and female (p < 0.05) rats compared to the respective control groups. (Figure-5 for males and figure-6 for females; Table-2). Table 2 Total neuron count and GFAP immunostaining in the hippocampal sections of study groups. Control group male rats Control group female rats Testosteron exposed male rats Testosteron exposed female rats CA1 Total neuron count 67.9 ± 5.5 66.5 ± 2.9 35.4 ± 5.1 ** 51.7 ± 2.5 * CA3 Total neuron count 45.4 ± 4.8 59.1 ± 4.3 28.7 ± 4.2 ** 30.8 ± 3.7 GFAP immunostaining CA1 21.6 ± 1.4 20.7 ± 1.5 33.2 ± 2.7 * 29.2 ± 1.9 * GFAP immunostaining CA3 19.1 ± 0.9 18.5 ± 1.2 27.1 ± 3.3 * 32.3 ± 1.2 * Results were presented as mean ± SEM. Statistical analyses were performed by one-way ANOVA. * p < 0.05, ** p < 0.001 different from control groups. Abbreviations: CA: Cornu Ammonis; GFAP: Glial fibrillary acidic protein. Discussion In this study, firstly, we found that chronic prenatal high-dose testosterone exposure caused autistic-like traits in rats, regardless of sex. Previous studies had shown that fetal testosterone exposure could lead to autistic traits in children (Auyeung et al., 2009; Auyeung et al., 2010; Hassan et al., 2019; James and Grech, 2020). Furthermore, Xu et al. found that testosterone levels were increased in the mothers of autistic children (Xu et al., 2013). Even though many studies have shown the effect of prenatal testosterone on autistic traits, the mechanism of effect of testosterone with respect to the development of autistic traits is unclear. We found that prenatal testosterone exposure leads to an increase in brain tissue 5- HVA levels. There are studies in the literature showing the effect of dopamine on autistic-like behaviors. Tyson et al. showed that testosterone could cause molecular changes in dopamine structure within the nigrostriatal pathway, thus increasing the effect of dopamine (Purves-Tyson et al., 2014). Putnam et al. showed that testosterone injections on castrated rats increased dopamine levels in the medial preoptic area which is an essential site for male sexual behavior (Putnam et al., 2001). Theije et al. showed that, in addition to an increase of dopamine levels in the amygdala, rats exposed to allergenic food demonstrated reduced social behavior and increased repetitive behaviors (de Theije et al., 2014). Our findings correlate with the previous studies and show the association between testosterone increase and autistic traits, possibly through the alteration of dopamine levels. Another striking result in our study is that prenatal testosterone exposure increases brain levels of 5-HIAA, the primary metabolite of serotonin. Previous studies have concluded that serotonin is one of the neurotransmitters that increases in patients with social disturbance and autistic children (Abdulamir et al., 2018; Naffah-Mazzacoratti et al., 1993; Yang et al., 2015). Dayem et al. found higher serum serotonin levels in autistic children compared to healthy controls, but also noted that there was no correlation with autism severity (Dayem H, 2018). In a similar study, Abdulamir et al. reported relatively elevated levels of serotonin and serotonin reuptake transporter (SERT) in autistic children, and their results demonstrated a correlation between serotonin level and autism severity (Abdulamir et al., 2018). Kranz et al. showed that high-dose testosterone treatment in female-to-male transgender people increased SERT binding in the brain, which may play a role in autistic traits (Kranz et al., 2015). Our findings appear to be well supported by the previous studies, and autism-like traits can be linked to serotonin increase as a result of prenatal testosterone exposure. The present study showed that prenatal testosterone exposure decreased oxytocin level in the rat brain. Oxytocin is a neurotransmitter that plays an essential role in social and affiliative behaviors. Research related to this role has uncovered that oxytocin administration may improve autistic traits in rats, and therefore, oxytocin has been considered a promising therapeutic agent for therapy in autistic trait behavior (Sala et al., 2011; Teng et al., 2016). Teng et al. showed the efficiency of subchronic oxytocin treatment to increase sociability and ameliorate repetitive stereotypic movements in rats (Teng et al., 2016). Procyshyn et al. showed that experimental empathy induction increases oxytocin levels and decreases testosterone levels in people, indicating an inverse relationship between oxytocin and testosterone (Procyshyn et al., 2020). In accordance with these data, our results have shown that high-dose prenatal testosterone exposure caused decreased oxytocin levels in both male and female gender, which may, in turn, indicate a relationship with autistic behavior. Another significant finding in our study is increased neuronal loss and astrogliosis on the CA1 and CA3 regions of the hippocampal sections among rats exposed to high-dose testosterone, which indicates neurodegeneration in both male and female offspring. This conclusion may be related to our biochemical findings as a result of several possible mechanisms. For instance, it is likely that prenatal testosterone exposure may cause neuronal inflammation via IGF-1 increase, leading to cytokine inhibition and glial activation, thereby resulting in decreased neuroinflammation (Riikonen, 2017). Another mechanism may be that testosterone exposure inhibits oxytocin, thus causing neurodegeneration and gliosis (Sunnetci et al., 2020). Besides this, increased metabolites of serotonin and dopamine can also cause neurodegeneration, consistent with the literature (Klein et al., 2019; Yang et al., 2015). We believe that the results of this study, taken together, indicate that prenatal testosterone exposure may cause autistic traits via increased levels of dopamine, testosterone, and IGF-1, and decreased levels of oxytocin. The present findings might help to understand the mechanism of prenatal high-dose testosterone exposure in the development of autistic traits. Further studies should be done to confirm our findings and reveal mechanistic associations. References (2011) In: th (ed) Guide for the Care and Use of Laboratory Animals. Washington (DC). Abdulamir HA, Abdul-Rasheed OF and Abdulghani EA. (2018) Serotonin and serotonin transporter levels in autistic children. Saudi Med J 39: 487-494. Anderson GM, Freedman DX, Cohen DJ, et al. (1987) Whole blood serotonin in autistic and normal subjects. J Child Psychol Psychiatry 28: 885-900. Auyeung B, Baron-Cohen S, Ashwin E, et al. (2009) Fetal testosterone and autistic traits. Br J Psychol 100: 1-22. Auyeung B, Taylor K, Hackett G, et al. (2010) Foetal testosterone and autistic traits in 18 to 24-month-old children. Mol Autism 1: 11. Becker KG. (2012) Male gender bias in autism and pediatric autoimmunity. Autism Res 5: 77-83. Bradford MM. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254. Dayem H BM, Ragheb D. . (2018) Study of Serum Level of Serotonin in Children with Autism Spectrum Disorder. EC Paediatrics : 11-18. de Theije CG, Wu J, Koelink PJ, et al. (2014) Autistic-like behavioural and neurochemical changes in a mouse model of food allergy. Behav Brain Res 261: 265-274. Ellegood J and Crawley JN. (2015) Behavioral and Neuroanatomical Phenotypes in Mouse Models of Autism. Neurotherapeutics 12: 521-533. Erbas O, Erdogan MA, Khalilnezhad A, et al. (2018) Neurobehavioral effects of long-term maternal fructose intake in rat offspring. Int J Dev Neurosci 69: 68-79. Ferri SL, Abel T and Brodkin ES. (2018) Sex Differences in Autism Spectrum Disorder: a Review. Curr Psychiatry Rep 20: 9. Hassan MH, Desoky T, Sakhr HM, et al. (2019) Possible Metabolic Alterations among Autistic Male Children: Clinical and Biochemical Approaches. J Mol Neurosci 67: 204-216. Insel TR. (2010) The challenge of translation in social neuroscience: a review of oxytocin, vasopressin, and affiliative behavior. Neuron 65: 768-779. James WH and Grech V. (2020) Potential explanations of behavioural and other differences and similarities between males and females with autism spectrum disorder. Early Hum Dev 140: 104863. Klein MO, Battagello DS, Cardoso AR, et al. (2019) Dopamine: Functions, Signaling, and Association with Neurological Diseases. Cell Mol Neurobiol 39: 31-59. Kranz GS, Wadsak W, Kaufmann U, et al. (2015) High-Dose Testosterone Treatment Increases Serotonin Transporter Binding in Transgender People. Biol Psychiatry 78: 525-533. Maenner MJ, Shaw KA, Baio J, et al. (2020) Prevalence of Autism Spectrum Disorder Among Children Aged 8 Years - Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2016. MMWR Surveill Summ 69: 1-12. Moy SS, Nadler JJ, Perez A, et al. (2004) Sociability and preference for social novelty in five inbred strains: an approach to assess autistic-like behavior in mice. Genes Brain Behav 3: 287-302. Naffah-Mazzacoratti MG, Rosenberg R, Fernandes MJ, et al. (1993) Serum serotonin levels of normal and autistic children. Braz J Med Biol Res 26: 309-317. Posar A, Resca F and Visconti P. (2015) Autism according to diagnostic and statistical manual of mental disorders 5(th) edition: The need for further improvements. J Pediatr Neurosci 10: 146-148. Procyshyn TL, Watson NV and Crespi BJ. (2020) Experimental empathy induction promotes oxytocin increases and testosterone decreases. Horm Behav 117: 104607. Purves-Tyson TD, Owens SJ, Double KL, et al. (2014) Testosterone induces molecular changes in dopamine signaling pathway molecules in the adolescent male rat nigrostriatal pathway. PLoS One 9: e91151. Putnam SK, Du J, Sato S, et al. (2001) Testosterone restoration of copulatory behavior correlates with medial preoptic dopamine release in castrated male rats. Horm Behav 39: 216-224. Riikonen R. (2017) Insulin-Like Growth Factors in the Pathogenesis of Neurological Diseases in Children. Int J Mol Sci 18. Sala M, Braida D, Lentini D, et al. (2011) Pharmacologic rescue of impaired cognitive flexibility, social deficits, increased aggression, and seizure susceptibility in oxytocin receptor null mice: a neurobehavioral model of autism. Biol Psychiatry 69: 875-882. Sunnetci E, Solmaz V and Erbas O. (2020) Chronic Oxytocin treatment has long lasting therapeutic potential in a rat model of neonatal hypercapnic-hypoxia injury, through enhanced GABAergic signaling and by reducing hippocampal gliosis with its anti-inflammatory feature. Peptides 135: 170398. Taylor MJ, Rosenqvist MA, Larsson H, et al. (2020) Etiology of Autism Spectrum Disorders and Autistic Traits Over Time. JAMA Psychiatry 77: 936-943. Teng BL, Nikolova VD, Riddick NV, et al. (2016) Reversal of social deficits by subchronic oxytocin in two autism mouse models. Neuropharmacology 105: 61-71. Xu XJ, Shou XJ, Li J, et al. (2013) Mothers of autistic children: lower plasma levels of oxytocin and Arg-vasopressin and a higher level of testosterone. PLoS One 8: e74849. Yang CJ, Liu CL, Sang B, et al. (2015) The combined role of serotonin and interleukin-6 as biomarker for autism. Neuroscience 284: 290-296. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About In Review Editorial Policies 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-182999","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":11332458,"identity":"382e1630-8470-434e-a493-e80caff3aa8f","order_by":0,"name":"Evrim Senkal","email":"","orcid":"","institution":"Medeniyet Üniversitesi ; Department of Pediatrics","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Evrim","middleName":"","lastName":"Senkal","suffix":""},{"id":11332459,"identity":"5f4cff23-98ee-4b28-8af5-7f51ba51120d","order_by":1,"name":"Umut Eryigit","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYJCCAwwGMKaBhRxY5AFhLSA9zCAtEsZgkQTCFsG0MEgkNoAofFr4pc8YHvxS8Cdx7Yz8gx9+FEikzw87/BBoi52cbgN2LZJ9OQaHZQwMErfdSGaW7DGQyN14O80AqCXZ2OwADhed4TE4LAHRwiDNANIyOwGk5UDiNhxa7JG0MP8Gakk3nJ3+Aa8WAx4eg4MfIFrYQLYkyEvn4LdF4gxbwWEGA2PjbWcem1kC/WK4QTqn4ECCAW6/8Pcwb/7444+c7LbjiY9v/PhjIy8/O33zhw8VdnK4tDAwcBgw86A4FazSAKtaKGB/wPgDmS/fgE/1KBgFo2AUjEQAACbqYZfqStjnAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-0360-4849","institution":"Dogal Saglik Clinic","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Umut","middleName":"","lastName":"Eryigit","suffix":""},{"id":11332460,"identity":"a5a8a1b4-8e6e-47b9-9778-dd4b65356235","order_by":2,"name":"Mehmet Fatih Bozkurt","email":"","orcid":"","institution":"Afyonkarahisar Kocatepe Universitesi, Faculty of Veterinary, Department of Pathology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"Fatih","lastName":"Bozkurt","suffix":""},{"id":11332461,"identity":"49d140bc-aa48-4474-815b-10742356c51e","order_by":3,"name":"Volkan Solmaz","email":"","orcid":"","institution":"Cologne University department of neurophysiology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Volkan","middleName":"","lastName":"Solmaz","suffix":""},{"id":11332462,"identity":"79ab1eed-89c2-4fb0-b552-b0c11ce45643","order_by":4,"name":"Oytun Erbas","email":"","orcid":"","institution":"Istanbul Bilim Universitesi: TC Demiroglu Bilim Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Oytun","middleName":"","lastName":"Erbas","suffix":""}],"badges":[],"createdAt":"2021-01-29 09:40:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-182999/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-182999/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":5901936,"identity":"3d434ef9-331e-4c35-8efb-ddbe8cccd853","added_by":"auto","created_at":"2021-02-12 16:02:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":196144,"visible":true,"origin":"","legend":"Graphical abstract","description":"","filename":"Fig1..jpg","url":"https://assets-eu.researchsquare.com/files/rs-182999/v1/1989e182bc5c9089930e1995.jpg"},{"id":5901939,"identity":"56325f2b-eb40-4b28-be54-6e862a833b50","added_by":"auto","created_at":"2021-02-12 16:02:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":75916,"visible":true,"origin":"","legend":"Behavioral analysis of female and male rats. Results were presented as mean ± SEM. Statistical analyses were performed by one-way ANOVA. * p \u003c 0.05, ** p \u003c 0.001 different from control groups.","description":"","filename":"Fig2..jpg","url":"https://assets-eu.researchsquare.com/files/rs-182999/v1/3017ebc500dcab9ac0dfa862.jpg"},{"id":5902210,"identity":"75193f79-db28-43f8-8440-06132d4a3e4e","added_by":"auto","created_at":"2021-02-12 16:08:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":472451,"visible":true,"origin":"","legend":"CA1 and CA3 regions of hippocampus. Cresyl violet stain x 40 magnification. A-B: Control Group Male Rats CA1 and CA3, Normal pyramidal neuron (blue arrows); C-D: Testosteron exposed male rats was neural body degeneration \u0026 decreased neural count and dysmorphological changes (red arrows) CA1 and CA3. (Scale bars for 1 cm = 50 μm).","description":"","filename":"Fig3..jpg","url":"https://assets-eu.researchsquare.com/files/rs-182999/v1/bd8c968ac4697b8492f241cb.jpg"},{"id":5902068,"identity":"c5e5e841-3057-4eae-b7f5-09580abf526f","added_by":"auto","created_at":"2021-02-12 16:05:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":474779,"visible":true,"origin":"","legend":"CA1 and CA3 regions of hippocampus. Cresyl violet stain x 40 magnification. A-B: Control Group female Rats CA1 and CA3 regions, Normal pyramidal neuron (blue arrows); C-D: Testosteron exposed female rats was neural body degeneration \u0026 decreased neural count and dysmorphological changes (red arrows) CA1 and CA3 regions. (Scale bars for 1 cm = 50 μm).","description":"","filename":"Fig4..jpg","url":"https://assets-eu.researchsquare.com/files/rs-182999/v1/7bb0b5b698f34bcb948ebdae.jpg"},{"id":5902070,"identity":"1d174839-2c9e-405b-b943-3cee5c274815","added_by":"auto","created_at":"2021-02-12 16:05:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":603413,"visible":true,"origin":"","legend":"CA1 and CA3 regionsof hippocampus x 40 magnification. Astrogliosis was characterized by GFAP immunostaining. A-B: Control Group Male Rats CA1 and CA3 regions; C-D: Testosteron exposed male rats was increased glial activity (Brown staining) CA1 and CA3 regions. (Scale bars for 1 cm = 50 μm).","description":"","filename":"Fig5..jpg","url":"https://assets-eu.researchsquare.com/files/rs-182999/v1/d165d274704bf5bc6c5a1f72.jpg"},{"id":5902069,"identity":"27a001a1-fa83-48f7-8e93-035ed17a8f35","added_by":"auto","created_at":"2021-02-12 16:05:52","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":657882,"visible":true,"origin":"","legend":"CA1 and CA3 regions of hippocampus x 40 magnification. Astrogliosis was characterized by GFAP immunostaining. a-b, Control Group Female Rats CA1 and CA3 regions, c-d, Testosteron exposed female rats was increased glial activity (Brown staining) CA1 and CA3 regions. (Scale bars for 1 cm = 50 μm).","description":"","filename":"Fig6..jpg","url":"https://assets-eu.researchsquare.com/files/rs-182999/v1/ea43ced4b53850f9107d7049.jpg"},{"id":13660604,"identity":"9ee499f7-2294-4837-aa4a-d0543cf07706","added_by":"auto","created_at":"2021-09-17 10:25:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1322793,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-182999/v1/5979fe74-ca6a-44f7-ab6a-9efc2895e61d.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eChronic Testosterone Administration During Pregnancy Causes Autistic-like Traits in Rat Offspring: Possible Relationships with Cerebral Oxytocin, Dopamine, Serotonin and IGF-1 Levels.\u003c/p\u003e","fulltext":[{"header":"Introduction","content":" \u003cp\u003eAutism spectrum disorder (ASD) is a neurodevelopmental disorder that affects communication and behavior. It is characterized by early-onset deficits in social and communication skills, restricted interests and repetitive body movements (Posar et al., 2015). Autism prevalence has been increasing in recent years (Maenner et al., 2020). The etiology of ASD is unknown, but it is thought that genetic and environmental factors come together in the development of ASD (Taylor et al., 2020). Current data demonstrates that boys are affected around three times more frequently than girls (Becker, 2012; Ferri et al., 2018). However, the mechanisms underlying male vulnerability are not well-known and remain understudied.\u003c/p\u003e \u003cp\u003eA higher incidence of ASD in males can in fact be among the features that could help to enlighten its etiology. For instance, intrauterine fetal testosterone exposure has gained some recognition as arguably the most popular hypothesis concerning the etiology of autism. Previous studies have suggested that higher fetal testosterone exposure can cause autism-like behaviors in males (Ferri et al., 2018). However, the mechanism between prenatal high dose testosterone exposure and autistic traits is not well documented. Recent studies showed that testosterone induces molecular changes in dopamine in the nigrostriatal pathway, thus increasing the effect of dopamine in the body (Purves-Tyson et al., 2014). It is well known that increased dopamine causes behavioral anomalies (Klein et al., 2019). Serotonin is another neurotransmitter that is shown to increase in autistic children (Anderson et al., 1987). Dysregulation of serotonin has been linked to behavioral anomalies (Hassan et al., 2019), and the latest studies have shown that there is a possible relationship between increased intrauterine testosterone levels and serotonin levels. Oxytocin is a famous molecule that appears to be involved in the neurobiology of autism development as per the results of recent studies. Oxytocin has been shown to modify synaptic plasticity and modulate social behaviors (Insel, 2010). In addition, empathy induction among individuals has been associated with increased oxytocin levels (Procyshyn et al., 2020). IGF-1 (Insulin-like growth factor-1), which reduces neuroinflammation by affecting cytokines and glial activation, is another hormone that can be increased in autism (Riikonen, 2017).\u003c/p\u003e \u003cp\u003eThis experimental study exposed pregnant rats to testosterone and assessed the results on the offspring via behavioral parameters, such as locomotor activity and anxiety-like behavior, using the open field test and social interaction using the three-chamber test. Additionally, we aimed to evaluate the possible association between prenatal testosterone exposure and other biochemical parameters, including 5-Hydroxyindoleacetic acid (5-HIAA) (serotonin's primary metabolite), homovanillic acid (HVA) (associated with dopamine levels in the brain), oxytocin, and IGF-1. We also evaluated the effect of prenatal testosterone exposure on the cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3) regions of the hippocampus via neuronal morphology, total neuron count, and GFAP immunoexpression.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003e The experimental procedures employed in the present study were approved by the Animal Ethics Committee (199545685/2). The rats used in the experiment were obtained from the Experimental Animal Laboratory of Bilim University. All experiments were carried out with respect to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (US) (2011).\u003c/p\u003e\u003cp\u003eTwelve female and four male Wistar adult rats (220\u0026thinsp;\u0026plusmn;\u0026thinsp;10 g) were included in the study. The rats were housed in plastic cages and maintained under standard conditions with 12-hour light/dark cycles at room temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and a humidity of 45\u0026ndash;60%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003eStudy design\u003c/h2\u003e\u003cp\u003eFemale rats were randomly distributed into Group 1 (control, n\u0026thinsp;=\u0026thinsp;6) and Group 2 (recipents of testosterone undecanoate, n\u0026thinsp;=\u0026thinsp;6). All animals were monitored daily for behavior and health conditions throughout the study. Female rats were caged with a fertile male (three female/one male) for 2\u0026ndash;3 days during the oestrus period. Mating was checked by looking at the white vaginal plaque in rats. Then, male rats were removed from the cages.\u003c/p\u003e\u003cp\u003eGroup 1 rats were given 1 ml/kg % 0.9 NaCl saline on the 10th day of pregnancy. Group 2 rats were given 250 mg/kg testosterone undecanoate (Nebido Ampul, Bayer, 250 mg/ml) on the 10th day of pregnancy. On the day of birth, to ensure standardized maternal care, litter size was reduced to 9 pups per dam. The dams were allowed to raise their own litters until weaning on postnatal day 21 (P21). On P21, forty littermates (10 male control, ten female control, ten male Testosterone-exposed, and ten female Testosterone-exposed) were randomly separated and housed in same-sex and same study group cages with \u003cem\u003ead libitum\u003c/em\u003e access to standard rodent chow and tap water. These animals underwent behavioral testing in adulthood on P50. All behavioral experiments were conducted between 10.00 AM and 3.00 PM. A summary of the study design is presented in Fig.\u0026nbsp;1.\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003eBehavioral tests\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section4\"\u003e\u003ch2\u003eThree- chamber sociability and social novelty test\u003c/h2\u003e\u003cp\u003eThe sociability test was performed as previously described with minor modifications (Ellegood and Crawley, 2015; Erbas et al., 2018; Moy et al., 2004). Briefly, a Plexiglas cage (40 cm x 90 cm x 40 cm) was divided into three equal regions (40 cm x 30 cm x 40 cm). On the first day, rats were allowed to habituate in the test cage for 5 min (pre-test session). Twenty-four hours later, to test sociability, a stranger rat was placed inside a small plastic cage with mesh-like holes in one side chamber and an empty cage in the third chamber. Then, the test rat was placed in the center chamber, and the time spent in each region by the test rat was recorded for 10 min (sociability test). The test rat was considered to be in the chamber when its head and two front paws entered the chamber. The field floor was then cleaned between each test with 70% alcohol and dried with a paper towel to remove any traces of olfactory stimuli from the previous rat.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section4\"\u003e\u003ch2\u003eOpen field test\u003c/h2\u003e\u003cp\u003eThe open-field test was conducted in an open-aired box with dimensions of 50 cm x 50 cm x 40 cm (Erbas et al., 2018). At the beginning of the test, rats were gently placed in the center of the box and freely explored the arena for 5 minutes. Then, each rat was observed for 5 min to evaluate spontaneous activity level. The total number of ambulation (number of floor divisions crossed with the four paws) were recorded. The field floor was then cleaned between each animal with a 70% alcohol-water solution and dried with a paper towel to remove olfactory cues.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eHippocampus histopathology\u003c/h2\u003e\u003cp\u003eThe CA1 and CA3 regions of the hippocampus were chosen as the target areas to be examined for hippocampus evaluation. Briefly, following behavioral tests, animals were euthanized, and their brains were removed and fixed in 10% formaldehyde (0.1 M phosphate buffer saline, PBS) for three days. Then, they were moved into 30% sucrose and stored at 4\u0026deg;C until infiltration was complete. The brains were cut coronally on a sliding microtome at 40 \u0026micro;m and mounted on gelatinized glass slides. Cresyl violet staining to quantify the number of surviving neurons were performed in six sections per studied group by an image analysis system (Image-Pro Express 1.4.5, Media Cybernetics, Inc. USA).\u003c/p\u003e\u003cp\u003eFor GFAP immunohistochemistry, brain sections were incubated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (10%) for 30 min to eliminate endogenous peroxidase activity and blocked with 10% normal goat serum (Invitrogen) for one hour at room temperature. Subsequently, sections were incubated in primary antibodies against GFAP (Abcam, Inc., MA, US; 1/1000) for 24 h at 4\u0026deg;C. Antibody detection was performed with the Histostain-Plus Bulk kit (Invitrogen) against rabbit IgG, and 3,3\u0026prime; diaminobenzidine (DAB) was used to visualize the final product. All sections were washed in PBS and photographed with an Olympus C-5050 digital camera mounted on the Olympus BX51 microscope. To calculate the GFAP immunostaining index, GFAP-positive cells were counted at 40X magnification in randomized Sect.\u0026nbsp;(3\u0026ndash;4) for each rat. All histopathological examinations were performed by the same investigator, who was blinded to the study groups.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eTissue biochemical analysis\u003c/h2\u003e\u003cp\u003eAfter decapitation, brains were rapidly removed and stored at -20\u0026deg;C until biochemical analyses. For tissue analysis, whole cerebral tissues were homogenized with a glass homogenizer in 5X volume of phosphate-buffered saline (pH, 7.4) and centrifuged at 5000 \u003cem\u003eg\u003c/em\u003e for 15 min. The supernatant was then collected, and total protein concentration in the brain homogenates was determined according to Bradford\u003cb\u003e'\u003c/b\u003es method using bovine serum albumin as standard (Bradford, 1976).\u003c/p\u003e\u003cp\u003eThe brain levels of IGF-1 (Shanghai LZ Biotech Co., Ltd.), oxytocin (Shanghai LZ Biotech Co., Ltd.), 5-HIAA (Shanghai LZ Biotech Co., Ltd.), HVA (Cusabio) in the tissue supernatants were measured using commercially available rat enzyme-linked immunosorbent assay (ELISA) kits. All samples from each animal were measured in duplicate according to the manufacturer\u003cb\u003e'\u003c/b\u003es guidelines.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical evaluation was performed using SPSS version 15.0 for Windows (SPSS Inc., Chicago, IL, USA). Shapiro-Wilk's W and Levene's tests were used to check normality of distribution and homogeneity of variance, respectively. The results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Resultant p-values of \u0026lt;\u0026thinsp;0.05 were accepted to demonstrate statistical significance.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n\u003ch2 class=\"Heading\"\u003eThree-Chamber and Open Field Test\u003c/h2\u003e\n\u003cp\u003eIn the three-chamber sociability and social novelty test, both male and female testosterone-exposed rats spent less time with the stranger rat than the male and female rats of the control group, indicating somewhat less social affiliation and more autism-like phenotype (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for males; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for females). Both male and female testosterone-exposed rats spent significantly less time in the center of the open field area, which suggests increased anxiety and less locomotor activity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for males ; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for females). Results are shown in Fig.\u0026nbsp;2.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eTissue Biochemical Analysis\u003c/h2\u003e\n\u003cp\u003eThe concentration of IGF-I, 5-HIAA and HVA measured from brain tissues were significantly higher in both male (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively) and female (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively) testosterone-exposed rats compared to control male and female rats. The concentration of oxytocin measured from brain tissues was significantly lower in both male (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and female (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) testosterone-exposed rats compared to the male and female rats of the control group (Table-1).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eiochemical results of groups\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e\u003cstrong\u003eControl group male rats\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e\u003cstrong\u003eControl group female rats\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e\u003cstrong\u003eTestosterone exposed male rats\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e\u003cstrong\u003eTestosterone exposed female rats\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"121\"\u003e\n\u003ch4\u003e\u003cem\u003eIGF-1 (U/mg protein)\u003c/em\u003e\u003c/h4\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e7.2 \u0026plusmn; 0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e6.3 \u0026plusmn; 0.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e24.8 \u0026plusmn; 5.5 **\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e19.5 \u0026plusmn; 3.8 **\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOxytocin (pg/mg protein)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e65.1 \u0026plusmn; 8.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e70.9 \u0026plusmn; 7.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e22.6 \u0026plusmn; 7.2**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e48.9 \u0026plusmn; 10.6 *\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e5-HIAA (\u0026micro;mol/mg protein)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e13.2 \u0026plusmn; 2.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e8.8 \u0026plusmn; 1.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e33.1 \u0026plusmn; 6.7 *\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e24.5 \u0026plusmn; 6.2 **\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHVA (ng/mg protein)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e0.73 \u0026plusmn; 0.09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003e0.68 \u0026plusmn; 0.04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e1.2 \u0026plusmn; 0.1 **\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003e0.9 \u0026plusmn; 0.3 *\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\" width=\"121\"\u003e\n\u003cp\u003eResults were presented as mean \u0026plusmn; SEM. Statistical analyses were performed by one-way ANOVA. * p \u0026lt; 0.05,\u0026nbsp; ** p \u0026lt; 0.001 different from control groups.\u003c/p\u003e\n\u003cp\u003eAbbreviations:\u0026nbsp; 5-HIAA:5-Hydroxyindoleacetic acid; HVA : Homovanillic acid; IGF-1: Insulin-like growth factor-1; SEM: standard\u0026nbsp;error of the mean.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eHistopathological evaluation of the groups\u003c/h2\u003e\n\u003cp\u003eDecreased neuronal count, and dysmorphological changes in both the CA1 and CA3 regions of the hippocampus, which indicates neuronal body degeneration, were present in both testosterone-exposed male (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and female (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) rats. In contrast, the cresyl violet staining results of control group rats demonstrated normal characteristics (Figure-3 for males and figure-4 for females). In both the CA1 and CA3 regions of the hippocampus, GFAP immunoreactivity of hippocampal sections showed increased glial activity in testosterone-exposed male (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and female (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) rats compared to the respective control groups. (Figure-5 for males and figure-6 for females; Table-2).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eTotal neuron count and GFAP immunostaining in the hippocampal sections of study groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e\u003cstrong\u003eControl group male rats\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e\u003cstrong\u003eControl group female rats\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e\u003cstrong\u003eTestosteron exposed male rats\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e\u003cstrong\u003eTestosteron exposed female rats\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCA1 Total neuron count\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e67.9 \u0026plusmn; 5.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e66.5 \u0026plusmn; 2.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e35.4 \u0026plusmn; 5.1 **\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e51.7 \u0026plusmn; 2.5 *\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCA3\u0026nbsp; Total neuron count\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e45.4 \u0026plusmn; 4.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e59.1 \u0026plusmn; 4.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e28.7 \u0026plusmn; 4.2 **\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e30.8 \u0026plusmn; 3.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGFAP immunostaining\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCA1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e21.6 \u0026plusmn; 1.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e20.7 \u0026plusmn; 1.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e33.2 \u0026plusmn; 2.7 *\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e29.2 \u0026plusmn; 1.9 *\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGFAP immunostaining\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCA3\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e19.1 \u0026plusmn; 0.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e18.5 \u0026plusmn; 1.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e27.1 \u0026plusmn; 3.3 *\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"122\"\u003e\n\u003cp\u003e32.3 \u0026plusmn; 1.2 *\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\" width=\"122\"\u003e\n\u003cp\u003eResults were presented as mean \u0026plusmn; SEM. Statistical analyses were performed by one-way ANOVA. * p \u0026lt; 0.05,\u0026nbsp; ** p \u0026lt; 0.001 different from control groups.\u003c/p\u003e\n\u003cp\u003eAbbreviations: CA: Cornu\u0026nbsp;Ammonis;\u0026nbsp; GFAP: Glial fibrillary\u0026nbsp;acidic protein.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, firstly, we found that chronic prenatal high-dose testosterone exposure caused autistic-like traits in rats, regardless of sex. Previous studies had shown that fetal testosterone exposure could lead to autistic traits in children (Auyeung et al., 2009; Auyeung et al., 2010; Hassan et al., 2019; James and Grech, 2020). Furthermore, Xu et al. found that testosterone levels were increased in the mothers of autistic children (Xu et al., 2013). Even though many studies have shown the effect of prenatal testosterone on autistic traits, the mechanism of effect of testosterone with respect to the development of autistic traits is unclear. We found that prenatal testosterone exposure leads to an increase in brain tissue 5- HVA levels. There are studies in the literature showing the effect of dopamine on autistic-like behaviors. Tyson et al. showed that testosterone could cause molecular changes in dopamine structure within the nigrostriatal pathway, thus increasing the effect of dopamine (Purves-Tyson et al., 2014). Putnam et al. showed that testosterone injections on castrated rats increased dopamine levels in the medial preoptic area which is an essential site for male sexual behavior (Putnam et al., 2001). Theije et al. showed that, in addition to an increase of dopamine levels in the amygdala, rats exposed to allergenic food demonstrated reduced social behavior and increased repetitive behaviors (de Theije et al., 2014). Our findings correlate with the previous studies and show the association between testosterone increase and autistic traits, possibly through the alteration of dopamine levels.\u003c/p\u003e\n\u003cp\u003eAnother striking result in our study is that prenatal testosterone exposure increases brain levels of 5-HIAA, the primary metabolite of serotonin. Previous studies have concluded that serotonin is one of the neurotransmitters that increases in patients with social disturbance and autistic children (Abdulamir et al., 2018; Naffah-Mazzacoratti et al., 1993; Yang et al., 2015). Dayem et al. found higher serum serotonin levels in autistic children compared to healthy controls, but also noted that there was no correlation with autism severity (Dayem H, 2018). In a similar study, Abdulamir et al. reported relatively elevated levels of serotonin and serotonin reuptake transporter (SERT) in autistic children, and their results demonstrated a correlation between serotonin level and autism severity (Abdulamir et al., 2018). Kranz et al. showed that high-dose testosterone treatment in female-to-male transgender people increased SERT binding in the brain, which may play a role in autistic traits (Kranz et al., 2015). Our findings appear to be well supported by the previous studies, and autism-like traits can be linked to serotonin increase as a result of prenatal testosterone exposure.\u003c/p\u003e\n\u003cp\u003eThe present study showed that prenatal testosterone exposure decreased oxytocin level in the rat brain. Oxytocin is a neurotransmitter that plays an essential role in social and affiliative behaviors. Research related to this role has uncovered that oxytocin administration may improve autistic traits in rats, and therefore, oxytocin has been considered a promising therapeutic agent for therapy in autistic trait behavior (Sala et al., 2011; Teng et al., 2016). Teng et al. showed the efficiency of subchronic oxytocin treatment to increase sociability and ameliorate repetitive stereotypic movements in rats (Teng et al., 2016). Procyshyn et al. showed that experimental empathy induction increases oxytocin levels and decreases testosterone levels in people, indicating an inverse relationship between oxytocin and testosterone (Procyshyn et al., 2020). In accordance with these data, our results have shown that high-dose prenatal testosterone exposure caused decreased oxytocin levels in both male and female gender, which may, in turn, indicate a relationship with autistic behavior.\u003c/p\u003e\n\u003cp\u003eAnother significant finding in our study is increased neuronal loss and astrogliosis on the CA1 and CA3 regions of the hippocampal sections among rats exposed to high-dose testosterone, which indicates neurodegeneration in both male and female offspring. This conclusion may be related to our biochemical findings as a result of several possible mechanisms. For instance, it is likely that prenatal testosterone exposure may cause neuronal inflammation via IGF-1 increase, leading to cytokine inhibition and glial activation, thereby resulting in decreased neuroinflammation (Riikonen, 2017). Another mechanism may be that testosterone exposure inhibits oxytocin, thus causing neurodegeneration and gliosis (Sunnetci et al., 2020). Besides this, increased metabolites of serotonin and dopamine can also cause neurodegeneration, consistent with the literature (Klein et al., 2019; Yang et al., 2015).\u003c/p\u003e\n\u003cp\u003eWe believe that the results of this study, taken together, indicate that prenatal testosterone exposure may cause autistic traits via increased levels of dopamine, testosterone, and IGF-1, and decreased levels of oxytocin. The present findings might help to understand the mechanism of prenatal high-dose testosterone exposure in the development of autistic traits. Further studies should be done to confirm our findings and reveal mechanistic associations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e(2011) In: th (ed) \u003cem\u003eGuide for the Care and Use of Laboratory Animals.\u003c/em\u003e Washington (DC).\u003c/li\u003e\n\u003cli\u003eAbdulamir HA, Abdul-Rasheed OF and Abdulghani EA. (2018) Serotonin and serotonin transporter levels in autistic children. \u003cem\u003eSaudi Med J\u003c/em\u003e 39: 487-494.\u003c/li\u003e\n\u003cli\u003eAnderson GM, Freedman DX, Cohen DJ, et al. (1987) Whole blood serotonin in autistic and normal subjects. \u003cem\u003eJ Child Psychol Psychiatry\u003c/em\u003e 28: 885-900.\u003c/li\u003e\n\u003cli\u003eAuyeung B, Baron-Cohen S, Ashwin E, et al. (2009) Fetal testosterone and autistic traits. \u003cem\u003eBr J Psychol\u003c/em\u003e 100: 1-22.\u003c/li\u003e\n\u003cli\u003eAuyeung B, Taylor K, Hackett G, et al. (2010) Foetal testosterone and autistic traits in 18 to 24-month-old children. \u003cem\u003eMol Autism\u003c/em\u003e 1: 11.\u003c/li\u003e\n\u003cli\u003eBecker KG. (2012) Male gender bias in autism and pediatric autoimmunity. \u003cem\u003eAutism Res\u003c/em\u003e 5: 77-83.\u003c/li\u003e\n\u003cli\u003eBradford MM. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. \u003cem\u003eAnal Biochem\u003c/em\u003e 72: 248-254.\u003c/li\u003e\n\u003cli\u003eDayem H BM, Ragheb D. . (2018) Study of Serum Level of Serotonin in Children with Autism Spectrum Disorder. \u003cem\u003eEC Paediatrics\u003c/em\u003e: 11-18.\u003c/li\u003e\n\u003cli\u003ede Theije CG, Wu J, Koelink PJ, et al. (2014) Autistic-like behavioural and neurochemical changes in a mouse model of food allergy. \u003cem\u003eBehav Brain Res\u003c/em\u003e 261: 265-274.\u003c/li\u003e\n\u003cli\u003eEllegood J and Crawley JN. (2015) Behavioral and Neuroanatomical Phenotypes in Mouse Models of Autism. \u003cem\u003eNeurotherapeutics\u003c/em\u003e 12: 521-533.\u003c/li\u003e\n\u003cli\u003eErbas O, Erdogan MA, Khalilnezhad A, et al. (2018) Neurobehavioral effects of long-term maternal fructose intake in rat offspring. \u003cem\u003eInt J Dev Neurosci\u003c/em\u003e 69: 68-79.\u003c/li\u003e\n\u003cli\u003eFerri SL, Abel T and Brodkin ES. (2018) Sex Differences in Autism Spectrum Disorder: a Review. \u003cem\u003eCurr Psychiatry Rep\u003c/em\u003e 20: 9.\u003c/li\u003e\n\u003cli\u003eHassan MH, Desoky T, Sakhr HM, et al. (2019) Possible Metabolic Alterations among Autistic Male Children: Clinical and Biochemical Approaches. \u003cem\u003eJ Mol Neurosci\u003c/em\u003e 67: 204-216.\u003c/li\u003e\n\u003cli\u003eInsel TR. (2010) The challenge of translation in social neuroscience: a review of oxytocin, vasopressin, and affiliative behavior. \u003cem\u003eNeuron\u003c/em\u003e 65: 768-779.\u003c/li\u003e\n\u003cli\u003eJames WH and Grech V. (2020) Potential explanations of behavioural and other differences and similarities between males and females with autism spectrum disorder. \u003cem\u003eEarly Hum Dev\u003c/em\u003e 140: 104863.\u003c/li\u003e\n\u003cli\u003eKlein MO, Battagello DS, Cardoso AR, et al. (2019) Dopamine: Functions, Signaling, and Association with Neurological Diseases. \u003cem\u003eCell Mol Neurobiol\u003c/em\u003e 39: 31-59.\u003c/li\u003e\n\u003cli\u003eKranz GS, Wadsak W, Kaufmann U, et al. (2015) High-Dose Testosterone Treatment Increases Serotonin Transporter Binding in Transgender People. \u003cem\u003eBiol Psychiatry\u003c/em\u003e 78: 525-533.\u003c/li\u003e\n\u003cli\u003eMaenner MJ, Shaw KA, Baio J, et al. (2020) Prevalence of Autism Spectrum Disorder Among Children Aged 8 Years - Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2016. \u003cem\u003eMMWR Surveill Summ\u003c/em\u003e 69: 1-12.\u003c/li\u003e\n\u003cli\u003eMoy SS, Nadler JJ, Perez A, et al. (2004) Sociability and preference for social novelty in five inbred strains: an approach to assess autistic-like behavior in mice. \u003cem\u003eGenes Brain Behav\u003c/em\u003e 3: 287-302.\u003c/li\u003e\n\u003cli\u003eNaffah-Mazzacoratti MG, Rosenberg R, Fernandes MJ, et al. (1993) Serum serotonin levels of normal and autistic children. \u003cem\u003eBraz J Med Biol Res\u003c/em\u003e 26: 309-317.\u003c/li\u003e\n\u003cli\u003ePosar A, Resca F and Visconti P. (2015) Autism according to diagnostic and statistical manual of mental disorders 5(th) edition: The need for further improvements. \u003cem\u003eJ Pediatr Neurosci\u003c/em\u003e 10: 146-148.\u003c/li\u003e\n\u003cli\u003eProcyshyn TL, Watson NV and Crespi BJ. (2020) Experimental empathy induction promotes oxytocin increases and testosterone decreases. \u003cem\u003eHorm Behav\u003c/em\u003e 117: 104607.\u003c/li\u003e\n\u003cli\u003ePurves-Tyson TD, Owens SJ, Double KL, et al. (2014) Testosterone induces molecular changes in dopamine signaling pathway molecules in the adolescent male rat nigrostriatal pathway. \u003cem\u003ePLoS One\u003c/em\u003e 9: e91151.\u003c/li\u003e\n\u003cli\u003ePutnam SK, Du J, Sato S, et al. (2001) Testosterone restoration of copulatory behavior correlates with medial preoptic dopamine release in castrated male rats. \u003cem\u003eHorm Behav\u003c/em\u003e 39: 216-224.\u003c/li\u003e\n\u003cli\u003eRiikonen R. (2017) Insulin-Like Growth Factors in the Pathogenesis of Neurological Diseases in Children. \u003cem\u003eInt J Mol Sci\u003c/em\u003e 18.\u003c/li\u003e\n\u003cli\u003eSala M, Braida D, Lentini D, et al. (2011) Pharmacologic rescue of impaired cognitive flexibility, social deficits, increased aggression, and seizure susceptibility in oxytocin receptor null mice: a neurobehavioral model of autism. \u003cem\u003eBiol Psychiatry\u003c/em\u003e 69: 875-882.\u003c/li\u003e\n\u003cli\u003eSunnetci E, Solmaz V and Erbas O. (2020) Chronic Oxytocin treatment has long lasting therapeutic potential in a rat model of neonatal hypercapnic-hypoxia injury, through enhanced GABAergic signaling and by reducing hippocampal gliosis with its anti-inflammatory feature. \u003cem\u003ePeptides\u003c/em\u003e 135: 170398.\u003c/li\u003e\n\u003cli\u003eTaylor MJ, Rosenqvist MA, Larsson H, et al. (2020) Etiology of Autism Spectrum Disorders and Autistic Traits Over Time. \u003cem\u003eJAMA Psychiatry\u003c/em\u003e 77: 936-943.\u003c/li\u003e\n\u003cli\u003eTeng BL, Nikolova VD, Riddick NV, et al. (2016) Reversal of social deficits by subchronic oxytocin in two autism mouse models. \u003cem\u003eNeuropharmacology\u003c/em\u003e 105: 61-71.\u003c/li\u003e\n\u003cli\u003eXu XJ, Shou XJ, Li J, et al. (2013) Mothers of autistic children: lower plasma levels of oxytocin and Arg-vasopressin and a higher level of testosterone. \u003cem\u003ePLoS One\u003c/em\u003e 8: e74849.\u003c/li\u003e\n\u003cli\u003eYang CJ, Liu CL, Sang B, et al. (2015) The combined role of serotonin and interleukin-6 as biomarker for autism. \u003cem\u003eNeuroscience\u003c/em\u003e 284: 290-296.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Autism spectrum disorder (ASD), neurodevelopmental disorder, higher fetal testosterone exposure","lastPublishedDoi":"10.21203/rs.3.rs-182999/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-182999/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects boys three times more frequently than girls. Previous studies have suggested that higher fetal testosterone exposure can cause autism-like behaviors in males. The mechanism of effect of fetal testosterone on autism development is not well known. In the present study, we investigated the relationship between prenatal testosterone exposure and ASD in an experimental study with the use of behavioral tests, histopathological examinations and biochemical measurements performed to compare offspring with and without exposure to testosterone.\nFemale rats were randomly distributed into Group 1 (control, n = 6) and Group 2 (Testosterone undecanoate, n = 6). Female rats were caged with a fertile male (three female/one male) for 2–3 days during the oestrus period.\nGroup 1 rats were given 1 ml/kg % 0.9 NaCl saline on the 10th day of pregnancy, while Group 2 rats were given 250 mg/kg testosterone undecanoate. The dams were allowed to raise their litters until weaning on postnatal day 21 (P21). On P21 , forty littermates (10 male control, ten female control, ten male Testosterone-exposed, and ten female Testosterone-exposed) were randomly separated and housed. These animals underwent behavioral testing in adulthood on P50. Subsequently, the rats were sacrificed. Biochemical analyses [Brain tissue 5-Hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), Insulin-like growth factor-1 (IGF-1), and oxytocin] and histopathological analyses were performed.\nAnalysis of the behavioral tests (three-chamber social test, open field test) revealed significant differences among the groups, with the testosterone-exposed groups demonstrating autistic traits at a greater degree. Histologically, hippocampal CA1 and CA3 regions displayed significant alterations such as gliosis and neuronal cell death in the testosterone-exposed groups compared to controls. Brain levels of tissue 5-HIAA, HVA, IGF-1 increased while oxytocin level decreased in the testosterone-exposed groups.\nThese results suggest a possible link between chronic prenatal testosterone exposure and neurodevelopmental disorders such as ASD. Testosterone exposure and autism-like traits can be linked to dopamine, serotonin, IGF-1 increase, and oxytocin decrease.","manuscriptTitle":"Chronic Testosterone Administration During Pregnancy Causes Autistic-like Traits in Rat Offspring: Possible Relationships with Cerebral Oxytocin, Dopamine, Serotonin and IGF-1 Levels.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-12 16:02:50","doi":"10.21203/rs.3.rs-182999/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":"b837382c-0e44-4bc8-b734-82d98221062d","owner":[],"postedDate":"February 12th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":2388622,"name":"Endocrinology \u0026 Metabolism"},{"id":2388623,"name":"Neurology"},{"id":2388624,"name":"Scientific Communication"},{"id":2388625,"name":"Developmental Neuroscience"}],"tags":[],"updatedAt":"2021-03-18T08:51:01+00:00","versionOfRecord":[],"versionCreatedAt":"2021-02-12 16:02:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-182999","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-182999","identity":"rs-182999","version":["v1"]},"buildId":"pf3fE39SIOqb-0xH_OWvX","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}