Sex Differences in Acute Neuroendocrine Responses to Stressors in Rodents and Humans

Abstract

Sex differences in the neuroendocrine response to acute stress occur in both animals and humans. In rodents, stressors such as restraint and novelty induce a greater activation of the hypothalamic-pituitary-adrenal axis (HPA) in females compared to males. The nature of this difference arises from steroid actions during development (organizational effects) and adulthood (activational effects). Androgens decrease HPA stress responsivity to acute stress, while estradiol increases it. Androgenic down-regulation of HPA responsiveness is mediated by the binding of testosterone (T) and dihydrotestosterone (DHT) to the androgen receptor, as well as the binding of the DHT metabolite, 3β-diol, to the β form of the estrogen receptor (ERβ). Estradiol binding to the α form of the estrogen receptor (ERα) increases HPA responsivity. Studies of human sex differences are relatively few and generally employ a psychosocial paradigm to measure stress-related HPA activation. Men consistently show greater HPA reactivity than women when being evaluated for achievement. Some studies have found greater reactivity in women when being evaluated for social performance. The pattern is inconsistent with rodent studies but may involve the differential nature of the stressors employed. Psychosocial stress is nonphysical and invokes a significant degree of top-down processing that is not easily comparable to the types of stressors employed in rodents. Gender identity may also be a factor based on recent work showing that it influences the neural processing of positive and negative emotional stimuli independent of genetic sex. Comparing different types of stressors and how they interact with gender identity and genetic sex will provide a better understanding of sex steroid influences on stress-related HPA reactivity.

Hans Selye (1950) introduced the term “stress” to describe a unidimensional reflex arc initiated by physiological and psychological factors that trigger endocrine and autonomic responses to threatening stimuli. This formed the basis of his General Adaptation Syndrome that emphasized the role of elevated levels of adrenal glucocorticoids (GCs) as an essential link between stress and medical illness. The reflexive nature of the response was later modified by studies in humans and primates showing that elevated cortisol levels were strongly related to the degree of situational control and anxiety, as opposed to overall physiological reactivity (Mason 1975). This perspective led to the view that neuroendocrine responses to stress reflect the impact of psychosocial factors versus arousal. Further modifications to the reflexive concept arose from studies demonstrating dissociations between elevated cortisol levels and self-report of anxiety or stress levels in humans due to individual differences, as well as differences between men and women (Kudielka and Kirschbaum 2005; Reschke-Hernández et al. 2017).

The current model emphasizes individual differences in psychological stress reactivity operating within a conceptual framework consisting of an integrated set of distinct physiological, emotional, and cognitive networks (Buigs and Van Eden 2000; Goldstein et al. 2010). The physiological networks include neuroendocrine and autonomic responses that increase glucose availability, regulate cardiovascular tone, and suppress gonadal hormone release and immune responses. Emotional networks include the limbic system and amygdala, which coordinate motivational state with reflexive behavioral responses related to defensive or aggressive behaviors. Cognitive networks include the prefrontal and cingulate cortices, which serve to focus selective attention on social context and planning strategies for restoring behavioral equilibrium. The integration of these networks resides in hypothalamic circuitry that coordinates the systemic response to stressors according to physiological needs and behavioral goals. The networks are similar in rodents and humans, but the evolutionary expansion of the human telencephalon provides the cognitive network a more dominant and flexible role in psychological stress reactivity.

The fundamental work on physiological and behavioral reactivity to stress comes from animal studies conducted over the past century. These have set the stage for translational studies of psychological stress reactivity in humans and its role in disease and mental disorders (Frankenhaeuser 1996; Cohen et al. 2007; Zorn et al. 2017). However, studies that compared physiological and behavioral stress reactivity in men and women were uncommon for most of the twentieth century (Kajantie and Phillips 2006). This omission largely stemmed from a general assumption that sex differences in human physiological responses to stress were small and their relationship to illness was unlikely to extend beyond reproductive systems.

Over the past 20 years, that assumption has been challenged by a growing literature showing a variety of nonreproductive medical problems that can be exacerbated by stress interacting with the genetic sex of the individual. Among these are susceptibility to cardiovascular and autoimmune diseases, as well as affective disorders such as anxiety and depression (Maeng and Milad 2015; Rubinow and Schmidt 2019). However, there is still a significant gap in our mechanistic understanding of how gonadal steroids interact with genetic sex in human stress (Shors 2016; Helpman et al. 2017).

This article first considers mechanisms regulating the activation and feedback inhibition of the hypothalamic-pituitary-adrenal axis (HPA) in rodents, with a focus on how steroids modulate this system to establish sex differences in adulthood and during development. The discussion is followed by the consideration of functional sex differences in human stress activity and general approaches currently used to study stress-related HPA activation.

REGULATION OF THE HPA: ANIMAL STUDIES IN RODENTS

Selye's pioneering work showing that the adrenal glands of female rats are larger than in males provided the first evidence suggesting a potential sex difference in the physiological stress response (Selye 1937). This sex difference was later correlated with a higher rate of basal and stress-induced adrenal GC secretion in females, an effect that is mediated by estrogen (Kitay 1961, 1964). It is now well established in rodents that females exhibit greater diurnal fluctuations in plasma GCs and greater GC secretion in response to physical or psychological stressors (Bielohuby et al. 2007; Goel et al. 2014; Zuloaga et al. 2020). The magnitude of this sex difference in rodents is remarkable, with basal and stress-reactive levels of corticosterone (CORT) up to 1.5 to 2 times greater in females than males (Aloisi et al. 1998).

In mammals, adrenal GC secretion is controlled by the hypothalamus. The HPA represents a cascade of neural and humoral signals driven by actual or perceived changes in the environment (Handa and Weiser 2014). In response to changes that may affect physiological homeostasis, the HPA is activated through groups of neuropeptide-expressing neurons located within the paraventricular nucleus (PVN) of the hypothalamus. Corticotropin-releasing hormone (CRH) expressing neurons, located in the parvocellular subdivisions of the PVN, are critical for this response. The release of CRH into the hypophyseal portal system stimulates the release of adrenocorticotropic hormone (ACTH) from anterior pituitary corticotrophs, which subsequently stimulates the adrenal cortex to produce and release GCs into the general circulation. Circulating GCs then feed back at the anterior pituitary, hypothalamus, and higher brain areas to negatively regulate further secretion and, importantly, to alter stress-related behaviors (Herman et al. 1998, 2012).

Functional Sex Differences in the HPA

Sex differences in HPA function have been consistently reported in the literature, and some of the mechanisms underlying these differences have now been identified. In rats, the ACTH and CORT response of females (Viau et al. 2005; Iwasaki-Sekino et al. 2009; Heck and Handa 2019a) is characterized by a greater and prolonged secretion of ACTH and GCs indicating both enhanced stimulus reactivity, as well as reduced negative feedback (Handa et al. 1994; Babb et al. 2013). Changes in negative feedback have been tracked to changes in gonadal steroid levels (Heck et al. 2020).

Importantly, the HPA response to stressors should not be considered detrimental as GC hormones act in a largely beneficial fashion in the short term. Acute rises in GCs augment physiological functions involved in the fight or flight reaction, enhance cognition, and limit functions unnecessary for an immediate stress response (e.g., reproduction, immune function, digestion) (Lupien et al. 2002; Charmandari et al. 2005; Yuen et al. 2009). In rats, gonadectomy (GDX) of males and females minimizes the sex difference in CORT secretion showing that the response is partially due to sex differences in circulating gonadal hormones (Heck and Handa 2019a). Moreover, hormone replacement to GDX animals reinstates the size of the sex difference found in intact animals (Heck and Handa 2019a). Thus, numerous studies have demonstrated that estradiol (E2) enhances, whereas testosterone (T) treatment inhibits HPA reactivity (Viau and Meaney 1996; Heck and Handa 2019b). However, some studies have found that E2 can reduce HPA reactivity, indicating the complexity of this response may be tied to other factors besides just hormone presence. Whether these contrasting results are due to hormone dose, type or length of treatment exposure, or duration of GDX has not yet been explored in detail. As a result, the mechanisms by which these hormones act to influence HPA function are not completely resolved (Viau et al. 1999; Handa et al. 2013; Oyola et al. 2016).

In rats, the most important hypothalamic releasing factors for pituitary ACTH secretion are CRH and arginine vasopressin (AVP). Both factors are potent ACTH secretagogues with AVP potentiating ACTH-releasing activity of CRH several-fold both in vivo and in vitro (Rivier and Vale 1983; Young et al. 2007). Moreover, females have significantly greater numbers of CRH-immunoreactive (CRH-ir) neuronal cell bodies within the PVN than do males (Stinnett et al. 2015), and also exhibit greater diurnal variation of CRH immunoreactivity in these neurons than males (Critchlow et al. 1963; Smith and Norman 1987; Handa and McGivern 2017). These morphological data are consistent with physiological data showing a greater stress-responsive activation of the HPA axis in females (Seale et al. 2004).

Studies have shown greater female expression of AVP and CRH mRNA in the PVN, and more ACTH precursor (POMC) in anterior pituitary following restraint compared to males (Viau et al. 2005; Babb et al. 2013). However, sex differences in AVP expression have been reported for other brain areas, such as the bed nucleus of the stria terminalis (BNST), lateral septum, medial preoptic area (MPOA), and amygdala (Rood and De Vries 2011; DiBenedictis et al. 2017), where the number of AVP neurons is much greater in males than females (de Vries et al. 2008). Given that AVP augments the actions of CRH at the anterior pituitary and can be coreleased with CRH at the median eminence, greater extra-PVN levels in males likely indicate that sex differences in AVP are related to behavioral effects of central AVP neurotransmission rather than HPA regulation.

Sex Differences in Negative Feedback Regulation of the HPA

The stimulatory limb of the HPA axis is kept under continuous check by feedback inhibition (Sapolsky et al. 2000). Negative feedback consists of GC sensitive inputs from a number of upstream regions including the hippocampus, BNST, prefrontal cortex, and others (Herman et al. 2012). One factor underlying sex differences in GC secretion involves the intensity of feedback inhibition. The negative feedback action of GCs is mediated by two corticosteroid receptor types: type I or mineralocorticoid receptor (MR) and type II or GC receptor (GR). Both receptor types reside within cells of the hypothalamus, hippocampus, and anterior pituitary gland, as well as other brain areas (Reul and de Kloet 1985). Both receptors appear to be involved in differing aspects of feedback regulation.

MRs possess a very high affinity for corticosteroids and, as a result, are predominantly occupied at basal levels of hormone secretion (Reul and de Kloet 1985). GRs, in contrast, exhibit an approximately 10-fold lower affinity for GCs and, therefore, are occupied following GC elevations (Reul and de Kloet 1985), such as after a stress-induced rise in GCs. Although MR has a higher affinity for corticosteroids than GR, these receptors function together to return stress-responsive elevations in corticosteroids to baseline (Goel et al. 2014). Assuming receptor number corresponds to hormone sensitivity, sex differences in the HPA may arise as a result of sex differences in receptor number, indicating greater negative feedback sensitivity and lower basal and stress-activated hormone secretion. Conversely, lower receptor levels correspond to weaker negative feedback and, thus, higher basal and stress-responsive hormone secretion (Kolber and Muglia 2009; Solomon et al. 2015). The concentration of GR and MR and the function of GR and MR are lower in females than males in the hypothalamus, hippocampus, and other brain regions (Solomon et al. 2015). This pattern is consistent with that of other tissues such as the thymus and liver (Herman et al. 2016). Given similar levels of hormone, GR- or MR-mediated functions (such as autoregulation of the receptor) are less sensitive to GC modulation in females than males (Endres et al. 1979), indicating that other factors modulate GC function.

E2 Modulation of the HPA

The stage of the estrous cycle affects the female neuroendocrine response to stress as reflected in the plasma levels of corticosterone. Basal and stress-responsive corticosteroid levels are highest on proestrus when E2 levels are correspondingly high (Viau and Meaney 1991; Herman et al. 2016; Heck and Handa 2019a). This may be a result of impaired negative feedback regulation (Heck and Handa 2019a). Hormone replacement studies show that E2 treatment of GDX female and male rats enhances basal and stress-responsive secretion of ACTH and corticosteroids (Seale et al. 2004; Lund et al. 2006; Figueriedo et al. 2007; Weiser and Handa 2009). The actual mechanism(s) whereby E2 enhances stress-responsive ACTH and CORT secretion is not completely resolved. At the hypothalamic level, increases in HPA activation could be due to increased stimulation of PVN neurons, or reductions in inhibitory tone (Heck and Handa 2019b). Such possibilities are suggested by data showing increased expression of stress-inducible c-fos mRNA in the PVN following E2-treatment (Larkin et al. 2010). The prolonged stress-responsive secretion of ACTH and corticosteroids, coupled with elevated baseline secretion of ACTH and corticosteroids in females following E2-treatment, also suggests reduced HPA negative feedback mechanisms. Experimental manipulation of CORT secretion by E2 shows a similar pattern, where CORT secretion is suppressed in GDX females administered by the synthetic GC, dexamethasone (de Souza et al. 2019). This suppressive effect of E2 may be mediated by reduced γ-aminobutyric acid (GABA)-ergic inhibition as GAD67-positive neurons express estrogen receptor α (ERα) in the peri-PVN region (Weiser and Handa 2009).

Androgen Modulation of HPA Function

In comparison to E2, a large body of literature suggests androgens exert mainly inhibitory actions on the HPA axis (Viau and Meaney 1996; Sheng et al. 2021b). GDX of adult male rats increases CORT and ACTH responses to stressors and, correspondingly, the expression of c-fos in PVN neurons (Handa et al. 2013; Rosinger et al. 2019). Hormone replacement in GDX male rats with T or the nonaromatizable androgen, dihydrotestosterone (DHT), returns stress-responsive CORT and ACTH back to the levels found in intact males (Lund et al. 2006; Williamson and Viau 2008). Treatment of GDX animals with DHT also inhibits the stress-induced rise in PVN c-fos mRNA demonstrating that T effects are independent of T aromatization to E2 (Williamson and Viau 2008). Importantly, inhibition of 5α reductase in males causes a rise in basal and postrestraint corticosteroid secretion (Handa et al. 2013; Heck and Handa 2019a).

Although androgens can inhibit HPA function and reduce CRH-ir in the PVN (Heck and Handa 2019a), androgen receptors (ARs) are not found in CRH or AVP neurons within the rodent PVN (Bingaman et al. 1994; Heck and Handa 2019b). ARs have been reported in the PVN, but these AR-ir neurons are located in subdivisions of the PVN that project to spinal cord and brainstem autonomic nuclei (Bingham et al. 2011; Heck and Handa 2019b). Consequently, the assumption is that AR regulation of the HPA occurs trans-synaptically. Local application of T to the BNST and MPOA inhibits HPA responses to stress in adult male rats (Williamson and Viau 2008). However, local application of DHT to the PVN of males shows that DHT can directly affect PVN neurons through a mechanism not involving AR, but rather through estrogen receptor β (ERβ) (Lund et al. 2006; Handa et al. 2009). Local PVN application of DHT effectively inhibited HPA function. Moreover, local application of 5α-androstane-3β, 17β-diol (3β-diol), a metabolite of DHT that binds ERβ, was as effective as DHT in inhibiting HPA function as was PVN application of ERβ-selective agonists (Lund et al. 2006). Consistent with this finding, the effects of both DHT and 3β-diol can be blocked by ER antagonists, but not an AR antagonist treatment (Lund et al. 2006). Because 3β-diol can bind ERβ with moderate affinity, these data support the hypothesis that DHT can be metabolized to 3β-diol, a ligand that preferentially binds ERβ, and that ERβ acts to inhibit HPA function. These effects of DHT metabolites on ERβ also occur in females (Kudwa et al. 2014), although circulating DHT levels are much lower in females than males (Fig. 1; Handelsman et al. 2018).

Figure 1.

Effects of testosterone (T) and its metabolites on hypothalamic-pituitary-adrenal (HPA) axis and behavioral stress responses. This figure describes enzymes involved in the conversion of T and its metabolites and predicted effects produced by binding androgen receptors (ARs), estrogen receptor α (ERα), estrogen receptor β (ERβ), and γ-aminobutyric acid (GABA) receptors. Binding of ARs or ERβ is expected to decrease the HPA axis and behavioral stress responses. In contrast, actions at ERα increase the HPA axis response to stress. Effects of 3α-diol on the HPA axis are currently unknown. (HSD) Hydroxysteroid dehydrogenase, (3α-diol) 5α-androstane-3α, 17β-diol, (3β-diol) 5α-androstane-3β, 17β-diol, (RL-HSD) 11-cis-retinol dehydrogenase-like 3α-HSD. (The figure and legend are reprinted from Zuloaga et al. 2020 under the terms of a Creative Commons Attribution 4.0 International License.)

Organizational Actions of Gonadal Steroids Underlying Sex-Biases in HPA Function

Exposure to varying levels of gonadal steroids during perinatal life programs the brain to set up sex biases in the HPA. Such “organizational” actions permanently modify the morphology and neural circuitry of the brain (Sheng et al. 2021a). In male rodents, there are two important surges of T during pre- and postnatal development that are crucial for defeminization and masculinization of the rat brain: during late gestation days 18–19 and 2–4 h after parturition (Corbier et al. 1978; McGivern et al. 1988). Male adult rats that were GDX at birth showed elevated stress-induced ACTH and CORT secretions, increased PVN c-Fos activation, and reduced AR expression in BNST and medial amygdala nucleus, similar to females. Neonatal T-replacement reversed these effects (Bingham and Viau 2008). Moreover, T treatment of neonatal female rats leads to decreased HPA reactivity in adulthood, suggesting organizational effects by gonadal steroids that persist into adulthood (Seale et al. 2005). Chen et al. (2014) used a novel knockout mouse model to demonstrate that male mice with a testicular feminization mutation (Tfm), due to a dysfunctional AR, exhibit a more female-like HPA response to stressors. Such data indicate that T's modulation of the HPA in adulthood is AR-dependent as demonstrated by earlier studies using other Tfm models (Zuloaga et al. 2008, 2011). However, T can also be converted to E2 by the aromatase enzyme, which appears to be important for the organizational actions of T on the HPA response to stress. Indeed, because neonatal castration affects adult behavioral and CORT responses to stress in Tfm rats (Zuloaga et al. 2011), which have a dysfunctional AR, these organizational effects of T cannot be mediated by AR. In support of a role for aromatization, Bingham et al. (2012) showed elevated stress-induced CORT secretions and increased PVN c-Fos expression in neonate males that were implanted with a slow-release capsule containing an aromatase inhibitor 12 h after birth. Such a response is more typical of females and, therefore, these data implicate an alternate, ER-mediated organizational effect of T on the masculinization of the HPA.

SEX DIFFERENCES IN STRESS-INDUCED ACTIVATION OF THE HPA: HUMAN STUDIES

The pattern of stress-induced HPA activation in men and women discussed below exhibits some notable differences from sex differences observed in animals (Donner and Lowry 2013). Before addressing these differences, it is important to note differences in the perceptual nature of psychological stress between rodents and humans. The induction of psychological stress in animals relies strongly on physical experience (Altemus 2006). This includes procedures that have a physical or reflexive behavioral basis, such as restraint, swimming, shock, or species-specific behaviors like the inherent avoidance of open spaces in rodents (Bangasser and Wicks 2017; Rincón-Cortés et al. 2019). In contrast, human studies of neuroendocrine stress reactivity generally employ psychosocial procedures that have no physical component. Cross-species comparisons of what constitutes “stress” is also complicated by the evolutionary expansion of the human prefrontal cortex, including its reciprocal connections with the anterior cingulate cortex and amygdala, which allows more complex socioemotional analysis of internal and external stimuli (McEwen et al. 2015). This appraisal system is also influenced by hormone level changes associated with the menstrual cycle (Goel et al. 2014). Few animal studies have examined stress reactivity across the estrous cycle. Thus, human stress studies of nonclinical populations are generally designed around complex socioemotional-type stressors that are not easily modeled in animals.

The first programmatic study of human sex differences in stress-induced HPA activation was initiated by Frankenhaeuser (1996) and colleagues more than 50 years ago. In both laboratory and real-world situations, they found that men exhibited greater cortisol and autonomic responses than women when the stress involved external evaluation related to social standing and/or academic achievement. Kirschbaum et al. (1992, 1999) later formalized this psychosocial approach by developing a standardized experimental procedure known as the Trier Social Stress Test (TSST). Today, the TSST is the most widely used method for studying sex differences in human stress reactivity (Allen et al. 2017).

In the TSST procedure, participants are given a 3-min period to prepare a short interview-type speech to be presented before an unresponsive audience of evaluators. This is followed by 5 min for presentation of the speech, at the end of which participants are surprised with a 5-min mental arithmetic test. Subjective stress level is assessed with self-reported measures of stress, task difficulty, and anxiety level. Neuroendocrine activation is generally measured by salivary cortisol levels and many studies also include heart rate as an indicator of autonomic reactivity. Whereas the TSST is widely viewed as a general measure of psychosocial stress, it is important to note that its approach is weighted toward inducing achievement-related stress because it includes cognitive skills that are evaluated in a social context.

TSST studies consistently demonstrate greater male HPA activation during the stress period compared to females (Kirschbaum et al. 1999; Kudielka and Kirschbaum 2005; Uhart et al. 2006; Stephens et al. 2016; Reschke-Hernández et al. 2017). Interestingly, males also exhibited greater HPA reactivity than females in anticipation of experiencing the task. This finding suggests the sex difference in stress-induced cortisol levels arises from input to the PVN from higher levels related to perceptual evaluation of the experience. A limited amount of data indicate that the sex-related pattern is not related to pituitary sensitivity to CRH as no sex differences in cortisol levels were found following CRH infusion under nonstress conditions or during physical exercise stress (Kirschbaum et al. 1999).

Although males consistently show higher cortisol responses in the TSST, the size of the sex difference can vary depending upon the phase of the menstrual cycle. Numerous studies have reported female cortisol responses are smaller during the follicular phase compared to the luteal phase, while a few have found no difference (Kajantie and Phillips 2006; Stevens and Hamann 2012). Oral contraceptives blunt the female cortisol response or have no effect (Kirschbaum et al. 1999; Bouma et al. 2009; Cornelisse et al. 2011; Klumbies et al. 2014; Barel et al. 2018). Overall, the impact of hormonal differences between cycle phases is variable across TSST studies, somewhat contradictory, and associated with relatively small sample sizes (Stevens and Hamann 2012). A clearer picture emerges from a study of 798 men and women by Herbison et al. (2016). During the stress period, male cortisol levels were higher than female levels during all phases of the menstrual cycle. The largest sex difference occurred in the follicular phase, with smaller or similar differences observed at ovulation and the luteal phase. This pattern suggests that elevated E and P levels enhance the perception of stress in women.

The different cortisol response pattern indicates that men and women do not perceive the psychosocial stress of the TSST in the same way. This inference is consistent with established sex differences in the overall behavioral response to psychosocial threat. On average, men are more likely to exhibit direct physical or verbal aggression when socially challenged, compared with the tendency of women to employ indirect measures related to social exclusion (Benenson et al. 2011). Thus, the achievement aspect of the TSST procedure may contribute more to the male experience of stress threat and HPA activation than to the female experience.

Stroud et al. (2002) provided some support for this interpretation in a well-controlled study that used two conditions to induce psychosocial stress. The first was an achievement situation conceptually analogous to the TSST. Participants were assessed for verbal and arithmetic skills after being told the purpose of the study was to examine the relationship between physiological responses and intelligence. To enhance psychosocial stress, two confederates were employed in the social assessment phase who knew most of the answers. This ensured that participants performed poorly. The second condition involved social rejection and used the same two confederates, where the participant and the confederates were told that the purpose of the study was to better understand how individuals get to know one another. They would discuss two different topics while the experimenter videotaped the interactions. The confederates were trained to use subtle social and verbal skills to socially exclude the participant over the course of the discussion. Female participants in both conditions were balanced for menstrual cycle phases. The results showed a marked interaction between sex and condition in the HPA response. In the achievement condition, there was a twofold rise in male cortisol levels compared to baseline but no change in female levels. In the social rejection condition, there was a twofold rise in female levels but no change in male levels. Interestingly, men and women rated both conditions equally stressful.

Wang et al. (2007) incorporated functional magnetic resonance imaging (fMRI) into a variation of the TSST to examine neural activation patterns. Low and high stress levels were induced by having participants perform simple and complicated mental arithmetic tasks while being evaluated by researchers. Performance of males and females was similar. However, males perceived the condition as more stressful than females, whereas females rated the same condition as significantly more difficult than males. In this study, no stress-induced sex differences were observed in heart rate or cortisol responses, which may reflect the modifications made to accommodate the fMRI. Nevertheless, robust sex differences were observed in activation of the cortical stress response network. Male activation of the right prefrontal cortex was increased compared to females, while activation of the left orbitofrontal cortex was suppressed. Females showed greater activation in limbic areas that included the hippocampus, insula, and anterior cingulate cortex. Sex differences in cortical activation patterns were large enough that a discriminant statistical analysis of male and female patterns correctly classified the sex of the participant with 94% accuracy.

Although HPA responsivity in the TSST is smaller in women, females show enhanced limbic activation in some areas compared to men when processing negative emotional stimuli. Stimuli associated with negative emotions induced greater amygdala activity in women in contrast to greater amygdala responses to positive emotions in men (Kogler et al. 2015). Some studies have not found this pattern, but lack of controls for menstrual cycle phase may be a factor (Schienle et al. 2005; Stevens and Hamann 2012; Seo et al. 2017). Overall, the differential pattern in cortical and limbic activity may reflect greater depth of emotional processing in women, which may also contribute to their higher level of social cognition (Kret and de Gelder 2012).

Goldstein et al. (2010) demonstrated E2's effect on stress-related neural activation using a within-subject design that used fMRI to scan neural activity of women at two phases of the menstrual cycle. Stress was induced by having participants view visual stimuli with established negative valence. Women were scanned in the early follicular phase when E2 levels are low, and again in the midcycle phase around the time of ovulation when E2 levels are high, but before the large rise of P during the mid- to late luteal phase. Men were included in the study for comparison. Results showed few sex differences in cortical activation when women were in the early follicular phase. However, during the midcycle phase, the neural response of women was significantly less than men in the limbic areas that included the amygdala, hippocampus, anterior cingulate gyrus, medial prefrontal cortex, and orbital prefrontal cortex.

Males and females differ in the resting state functional connectivity of the basolateral amygdala, the region that stimulates fear responses in rats and humans, suggesting that the difference in the response to emotional stimuli is inherent. Females show greater basolateral connections with lateral frontal and striatal regions, while males show greater connectivity with medial frontal regions (Engman et al. 2016; McGlade et al. 2020). This pattern indicates that females have a hormonal capacity to regulate the stress response differently from males, resulting in a more efficient and effective connectivity that translates stress responses to subjective awareness (Ordaz and Luna 2012).

Because of the historical exclusion of sex in studies of stress reactivity, especially in nonclinical populations (Zucker et al. 2021), the current literature is limited, and leaves open important questions related to the broader issue of individual differences, some of which involve the definition of sex in research. It is generally understood that “sex” refers to sex chromosomes, while the term “gender” is defined by sociocultural expectations of behaviors and can denote a range of identities that do not have to correspond to established ideas of male and female based on sex chromosomes. Yet, the terms sex and gender are often used interchangeably in scientific papers and in studies of sex differences. The human studies cited in this chapter classified participants on the basis of genetic sex, leaving open the question of whether gender identification is a superseding influence on stress reactivity that is distinct from genetic sex.

Yuan et al. (2021) recently addressed part of this question in a study of men and women that included the constructs of gender and sex as independent variables. They examined selective attention using event-related potentials (ERPs) associated with visual neural processing of neutral stimuli as well as positive and negative stimuli of varying levels of emotional intensity. They employed an “oddball paradigm” with an infrequent standardized neutral stimulus shown randomly. Participants responded on each trial as to whether the stimulus matched the standardized neutral stimulus. Each participant was assessed for their gender identity, with their “masculine” and “feminine” profile scores included as an independent factor in addition to biological sex. Gender profiles were derived from a Chinese Sex Role Inventory modeled on the original Bem Sex Role Inventory (Bem 1981; Keyes 1984; Qian et al. 2000). The ERP response to both positive and negative stimuli revealed a significantly larger P100 component in both hemispheres for individuals with feminine gender roles compared to masculine. This effect was not observed when individuals were compared by genetic sex. Participants classified as feminine, regardless of biological sex, exhibited greater sensitivity to the valence of both positive and negative stimuli compared to participants classified as masculine. This feminine classification was associated with significantly longer reaction times to discriminate the standardized neutral stimulus compared to those classified as masculine. Biological males and females showed no sex difference in reaction time. A better understanding of steroid regulation of human HPA reactivity will emerge from future studies that incorporate gender identification as a defining variable in addition to genetic sex.

CONCLUSIONS

Studies of sex differences in the acute neuroendocrine response of rodents to stress show a role for both organizational and activational effects of gonadal steroid actions that regulate the HPA. Organizational effects of androgens induce a long-term decrease in HPA stress reactivity. Activational effects of androgens generally down-regulate HPA stress sensitivity in the rodent, whereas estrogens increase it. Androgenic down-regulation involves T and DHT binding to the AR, as well as the DHT metabolite, 3β-diol, binding to ERβ. Estrogen stimulation of HPA sensitivity is mediated through E2 binding to ERα. Sex differences are also found in hypothalamic expression of CRH and AVP, as well the concentration of GRs in brain areas that regulate HPA feedback.

Much less is known about the mechanisms of sex steroid regulation of HPA activity in humans. Men and women also exhibit differences in HPA reactivity following acute psychological stress, but the pattern differs from rodents. The species difference may reflect a lack of experimental equivalency between the stressors used in rodents versus the psychosocial stressors employed in men and women.

Understanding differences between men and women in their neuroendocrine stress responses is obviously a complex process and likely involves individual differences operating at multiple levels. Currently, our limited knowledge does not provide a clear path toward explaining how these differences relate to sex differences observed in stress-related clinical disorders. The task is growing more complex with the recognition of a role for gender and personality as additional factors associated with differences in structural and functional connectivity in brain regions that include stress networks (Burke et al. 2017; Nostro et al. 2017; Xin et al. 2017). Future studies integrating these factors into studies of hormonal and genetic influences on stress responsiveness in preclinical and clinical populations will be important for developing translational approaches to understanding and treating stress-related mental disorders and diseases.