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.