**5. Sex differences in responses to stress**

Men and women differ in the prevalence of chronic diseases. For example, men have a higher risk of infectious disease [51] and incidence of cardiovascular disease than women [52, 53] whereas women have a higher incidence of major depression and anxiety [54-56] and autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis [57] than men. Since there are also sex differences in the response to stress, the response to stress poses a potential candidate in the etiology of the chronic disease progression. As indicated above, we will focus on the sympatho-adrenal medullary system and hypothalamo-pituitary adrenal axis when considering sex differences in response to stress.

There has been relatively little research on sex differences in the response of the sympathoadrenal medullary system to stress compared to the hypothalamo-pituitary adrenal axis, where most of the effort has been concentrated. We conducted one study comparing plasma catecholamine concentrations in gonadectomized sheep subjected to isolation and restraint

Sex Differences and the Role of Sex Steroids in Sympatho-Adrenal

black bar). From [61].

Medullary System and Hypothalamo-Pituitary Adrenal Axis Responses to Stress 123

Fig. 2. Mean (±SEM) plasma concentrations of cortisol in gonadectomized male and female sheep before and during exposure to 180 min of isolation/restraint stress (indicated by the

Fig. 3. Mean (±SEM) plasma concentrations of cortisol in gonadectomized male and female sheep before, during and after exposure to 5 min of audiovisual stress (barking dog;

Fig. 4. Mean (±SEM) plasma concentrations of cortisol in gonadectomized male and female sheep before, during and after exposure to 30 min of wetting stress (indicated by the grey

indicated by the grey bar). From [62]. *Copyright S. Karger AG, Basel* 

bar). From [63]. *Copyright 2010, The Endocrine Society.* 

stress [58]. Plasma concentrations of epinephrine were significantly elevated above pretreatment concentrations for longer in rams (2-180 minutes after the commencement of stress) than in ewes (2-25 minutes after the commencement of stress). Nevertheless, there were no consistent significant differences between rams and ewes in plasma concentrations of epinephrine [58]. Interestingly, plasma concentrations of norepinephrine were not influenced by the isolation/restraint stress in either sex [58]. The reasons for this are not apparent given that the source of both catecholamines in plasma would have been the adrenal medulla.

In humans, the issue of sex differences in responses of the sympatho-adrenal medullary system to stress has been extensively reviewed [49] and sex differences are evident, although they do not always appear consistent. One reason for this is that physiological state influence these responses [49] and only a small number of studies have standardized the stage of the menstrual cycle thereby standardizing the sex steroid milieu of females. For example, one study reported that women in both the luteal and follicular phases of the menstrual cycle had a higher increase in heart rate in response to a psychological stress (mental arithmetic) compared to men but this sex difference was not present for a physical stress (cold pressor test) [59]. Women also tended to have a higher increase in diastolic blood pressure following the mental arithmetic stress compared to men (p<0.07) but, again, this was not the case for the physical stress and there were no differences between the sexes for systolic blood pressure [59]. In a different study, it was found that heart rate response to a psychosocial stress (Trier Social Stress Test) was significantly higher in luteal phase women compared to men [60]. Collectively, these findings indicate that sex differences in response of the sympatho-adrenal medullary system to stress will vary with different stressors, and this has also been apparent in various other studies [49]. This is not surprising and is also the case with respect to the hypothalamo-pituitary adrenal axis (see below). Furthermore, sex differences in the response of the sympatho-adrenal medullary system to stress in humans are influenced by age and reproductive hormonal status of the women [49].

We have demonstrated sex differences in the responsiveness of the hypothalamo-pituitary adrenal axis to stress in sheep. This was quantified on the basis of plasma concentrations of cortisol and it is evident that sex differences in responses to stress vary with the stressor. For instance, female sheep had a greater cortisol response to isolation/restraint stress (Figure 2)[61], an audiovisual stress (Figure 3)[62] and a wetting stress (Figure 4)[63] compared with male sheep, whereas male sheep had a greater cortisol response to insulin induced hypoglycaemia compared with female sheep (Figure 5) [61]. It is tempting to speculate that the direction of differences between females and males in cortisol responses to stress may be explained on the basis of psychosocial compared to metabolic stressors but further research is required to ascertain this. Besides, not all stressors elicit sex differences in cortisol responses, there being no differences between the sexes in the cortisol response to exercise stress (Figure 6) [63] or to endotoxin (Figure 7) [63]. Nevertheless, when sex differences do occur in sheep, it appears that the mechanisms for this are in place early in life, at least for some stressors. In lambs, we found that females had a significantly higher cortisol response to tail docking compared to males and that this sex difference developed between one and eight weeks of age [64]. We have also investigated the mechanisms for sex differences in hypothalamo-pituitary adrenal axis responses to stress and have found various differences between males and females at each level of the axis, some of which depend on gonadal factors [65, 66], which are discussed in the next section. These include differences in neuropeptide distribution in the paraventricular nucleus of the hypothalamus [66] as well as adrenal size and adrenal responsiveness to ACTH [65].

stress [58]. Plasma concentrations of epinephrine were significantly elevated above pretreatment concentrations for longer in rams (2-180 minutes after the commencement of stress) than in ewes (2-25 minutes after the commencement of stress). Nevertheless, there were no consistent significant differences between rams and ewes in plasma concentrations of epinephrine [58]. Interestingly, plasma concentrations of norepinephrine were not influenced by the isolation/restraint stress in either sex [58]. The reasons for this are not apparent given that the source of both catecholamines in plasma would have been the adrenal medulla. In humans, the issue of sex differences in responses of the sympatho-adrenal medullary system to stress has been extensively reviewed [49] and sex differences are evident, although they do not always appear consistent. One reason for this is that physiological state influence these responses [49] and only a small number of studies have standardized the stage of the menstrual cycle thereby standardizing the sex steroid milieu of females. For example, one study reported that women in both the luteal and follicular phases of the menstrual cycle had a higher increase in heart rate in response to a psychological stress (mental arithmetic) compared to men but this sex difference was not present for a physical stress (cold pressor test) [59]. Women also tended to have a higher increase in diastolic blood pressure following the mental arithmetic stress compared to men (p<0.07) but, again, this was not the case for the physical stress and there were no differences between the sexes for systolic blood pressure [59]. In a different study, it was found that heart rate response to a psychosocial stress (Trier Social Stress Test) was significantly higher in luteal phase women compared to men [60]. Collectively, these findings indicate that sex differences in response of the sympatho-adrenal medullary system to stress will vary with different stressors, and this has also been apparent in various other studies [49]. This is not surprising and is also the case with respect to the hypothalamo-pituitary adrenal axis (see below). Furthermore, sex differences in the response of the sympatho-adrenal medullary system to stress in humans are

influenced by age and reproductive hormonal status of the women [49].

adrenal size and adrenal responsiveness to ACTH [65].

We have demonstrated sex differences in the responsiveness of the hypothalamo-pituitary adrenal axis to stress in sheep. This was quantified on the basis of plasma concentrations of cortisol and it is evident that sex differences in responses to stress vary with the stressor. For instance, female sheep had a greater cortisol response to isolation/restraint stress (Figure 2)[61], an audiovisual stress (Figure 3)[62] and a wetting stress (Figure 4)[63] compared with male sheep, whereas male sheep had a greater cortisol response to insulin induced hypoglycaemia compared with female sheep (Figure 5) [61]. It is tempting to speculate that the direction of differences between females and males in cortisol responses to stress may be explained on the basis of psychosocial compared to metabolic stressors but further research is required to ascertain this. Besides, not all stressors elicit sex differences in cortisol responses, there being no differences between the sexes in the cortisol response to exercise stress (Figure 6) [63] or to endotoxin (Figure 7) [63]. Nevertheless, when sex differences do occur in sheep, it appears that the mechanisms for this are in place early in life, at least for some stressors. In lambs, we found that females had a significantly higher cortisol response to tail docking compared to males and that this sex difference developed between one and eight weeks of age [64]. We have also investigated the mechanisms for sex differences in hypothalamo-pituitary adrenal axis responses to stress and have found various differences between males and females at each level of the axis, some of which depend on gonadal factors [65, 66], which are discussed in the next section. These include differences in neuropeptide distribution in the paraventricular nucleus of the hypothalamus [66] as well as

Fig. 2. Mean (±SEM) plasma concentrations of cortisol in gonadectomized male and female sheep before and during exposure to 180 min of isolation/restraint stress (indicated by the black bar). From [61].

Fig. 3. Mean (±SEM) plasma concentrations of cortisol in gonadectomized male and female sheep before, during and after exposure to 5 min of audiovisual stress (barking dog; indicated by the grey bar). From [62]. *Copyright S. Karger AG, Basel* 

Fig. 4. Mean (±SEM) plasma concentrations of cortisol in gonadectomized male and female sheep before, during and after exposure to 30 min of wetting stress (indicated by the grey bar). From [63]. *Copyright 2010, The Endocrine Society.* 

Sex Differences and the Role of Sex Steroids in Sympatho-Adrenal

repeated stress, to ascertain the relative impact of stress on each sex.

**6. The role of sex steroids and reproductive state on stress responsiveness**  It is apparent that there are interactions between the stress systems and sex steroid producing systems. This is most marked with the interactions between the hypothalamopituitary adrenal axis and reproductive axis, which are bidirectional. Activation of the stress systems can impact the reproductive axis [16, 70-75] and sex steroids can affect activation of the stress systems. When it comes to trying to appreciate the role of sex steroids in influencing the stress systems one is compelled to consider research in rodents because this is where the majority of investigation has been. Furthermore, most research concerning the effects of steroids on stress systems has concentrated on the hypothalamo-pituitary adrenal axis, with relatively little attention paid to the sympatho-adrenal medullary system. Nevertheless, there are actions of sex steroids on catecholaminergic neurons in various brain

Medullary System and Hypothalamo-Pituitary Adrenal Axis Responses to Stress 125

In humans, there is also evidence that hypothalamo-pituitary adrenal axis responses to stress differ between adult men and women (see [49] for an extensive review of the literature). The initial research effort in humans was hampered by a lack of treatments that adequately activated the hypothalamo-pituitary adrenal axis [49]. More recent research has shown that there are only subtle sex differences in the basal activity of the hypothalamopituitary adrenal axis but these become more pronounced with the imposition of a psychological stressor. In general, it seems that between puberty and menopause, cortisol responses to psychosocial stress are lower in women compared with aged matched men [49, 67]. Nevertheless, as in our sheep studies, there is also some evidence from human studies that sex differences in cortisol responses to stress may depend on the stressor encountered since men had significantly greater cortisol responses to achievement challenges than women and women had significantly greater cortisol responses to social rejection challenges than men [49]. In contrast to studies in post-pubertal humans, few studies have found sex differences in stress responsiveness during infancy and childhood [49]. Unlike sheep where a window of development of sex differences has been identified [64], the precise stage of development of sex differences is unknown in humans. Nonetheless, there is a prolonged activation of the hypothalamo-pituitary adrenal axis to stress during adolescence (for review see [68, 69]). While there has been various research across a range of species to try and understand the mechanisms for sex differences in responses to stress, with a large emphasis on the role of gonadal factors such as the sex steroids (see Section 6), there has been little attention paid to understanding the physiological importance of these sex differences. This needs to be considered from both an adaptive perspective, and from consideration of the impact of stress on health. With regard to the former, if one considers that stress responses are designed to re-establish homeostasis, to ward off the detrimental effects of noxious stimuli (Section 2), then one could argue that the sex with the greater catecholamine and cortisol responses to a particular stressor is the better equipped to deal with the stress. On the other hand, if one considers that prolonged or repeated activation of stress systems can be damaging to health (Section 3), then the sex with the greater response may be in the greater danger of the deleterious effects of the response. Unfortunately, these hypotheses have hitherto not been tested and this highlights an important issue requiring research. There is a need to determine the salience of stressors when undertaking sex comparisons, and there is a need to undertake sex comparisons over extended periods, and under conditions of

Fig. 5. Mean (±SEM) plasma concentrations of cortisol in gonadectomized male and female sheep before and after injection of insulin (indicated by the arrow). From [61].

Fig. 6. Mean (±SEM) plasma concentrations of cortisol in gonadectomized male and female sheep before, during and after exercise stress, which consisted of running 3 x 0.6 km (indicated by the grey bars). From [63]. *Copyright 2010, The Endocrine Society.* 

Fig. 7. Mean (±SEM) plasma concentrations of cortisol in gonadectomized male and female sheep before, during and after injection of endotoxin (indicated by the arrow). From [63]. *Copyright 2010, The Endocrine Society.*

Fig. 5. Mean (±SEM) plasma concentrations of cortisol in gonadectomized male and female

Fig. 6. Mean (±SEM) plasma concentrations of cortisol in gonadectomized male and female sheep before, during and after exercise stress, which consisted of running 3 x 0.6 km (indicated

Fig. 7. Mean (±SEM) plasma concentrations of cortisol in gonadectomized male and female sheep before, during and after injection of endotoxin (indicated by the arrow). From [63].

by the grey bars). From [63]. *Copyright 2010, The Endocrine Society.* 

*Copyright 2010, The Endocrine Society.*

sheep before and after injection of insulin (indicated by the arrow). From [61].

In humans, there is also evidence that hypothalamo-pituitary adrenal axis responses to stress differ between adult men and women (see [49] for an extensive review of the literature). The initial research effort in humans was hampered by a lack of treatments that adequately activated the hypothalamo-pituitary adrenal axis [49]. More recent research has shown that there are only subtle sex differences in the basal activity of the hypothalamopituitary adrenal axis but these become more pronounced with the imposition of a psychological stressor. In general, it seems that between puberty and menopause, cortisol responses to psychosocial stress are lower in women compared with aged matched men [49, 67]. Nevertheless, as in our sheep studies, there is also some evidence from human studies that sex differences in cortisol responses to stress may depend on the stressor encountered since men had significantly greater cortisol responses to achievement challenges than women and women had significantly greater cortisol responses to social rejection challenges than men [49]. In contrast to studies in post-pubertal humans, few studies have found sex differences in stress responsiveness during infancy and childhood [49]. Unlike sheep where a window of development of sex differences has been identified [64], the precise stage of development of sex differences is unknown in humans. Nonetheless, there is a prolonged activation of the hypothalamo-pituitary adrenal axis to stress during adolescence (for review see [68, 69]).

While there has been various research across a range of species to try and understand the mechanisms for sex differences in responses to stress, with a large emphasis on the role of gonadal factors such as the sex steroids (see Section 6), there has been little attention paid to understanding the physiological importance of these sex differences. This needs to be considered from both an adaptive perspective, and from consideration of the impact of stress on health. With regard to the former, if one considers that stress responses are designed to re-establish homeostasis, to ward off the detrimental effects of noxious stimuli (Section 2), then one could argue that the sex with the greater catecholamine and cortisol responses to a particular stressor is the better equipped to deal with the stress. On the other hand, if one considers that prolonged or repeated activation of stress systems can be damaging to health (Section 3), then the sex with the greater response may be in the greater danger of the deleterious effects of the response. Unfortunately, these hypotheses have hitherto not been tested and this highlights an important issue requiring research. There is a need to determine the salience of stressors when undertaking sex comparisons, and there is a need to undertake sex comparisons over extended periods, and under conditions of repeated stress, to ascertain the relative impact of stress on each sex.
