**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

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

different stressors.

Medullary System and Hypothalamo-Pituitary Adrenal Axis Responses to Stress 127

Gonadectomy also elevated median eminence content of CRH but there was no difference between sexes. There was no effect of *in vitro* ACTH secretion in response to treatment with AVP, CRH and the two in combination [99]. Our research in prepubertal sheep has also shown that some sex differences in the activity of the hypothalamo-pituitary adrenal axis endure in the absence of the sex steroids [64]. Consistent with these findings, we also showed that sex and gonadal status affect the distribution of CRH, AVP and enkephalin in the paraventricular nucleus of sheep, providing a neuroanatomical basis for sex differences in the central regulation of the hypothalamo-pituitary adrenal axis [66]. Others have shown that steroids and reproductive state can affect the activity of the hypothalamo-pituitary adrenal axis in female sheep [100], but there were no comparisons with males. In a series of studies in ewes it was shown that progesterone inhibits ACTH stimulation of cortisol [101, 102, 103] and stress, induced secretion of cortisol estrogens and androgens influence hypothalamic concentrations of CRH and AVP [104], and that ovariectomy affects hypothalamo-pituitary adrenal axis responses to stress [105, 106]. While it is clear that gonadal factors, such as the sex steroids, and reproductive state, influence the activity of the hypothalamo-pituitary adrenal axis in sheep, there is a need for more extensive research to establish the precise roles of steroids and reproductive state, including with regard to

In women the stage of the reproductive cycle has been shown to affect the hypothalamopituitary adrenal axis responses to stress (for an extensive review see [49]). It has been consistently reported that cortisol responses to psychosocial stress were lower in women in the follicular phase of the menstrual cycle compared with women in the luteal phase of the menstrual cycle [49]. This suggests that there is attenuation of the hypothalamo-pituitary adrenal axis in response to psychosocial stress in an estrogenic environment (i.e. the follicular phase), and this is supported by studies showing that women taking a synthetic estrogen as an oral contraceptive had similar cortisol responses to psychosocial stress compared to women in the follicular phase of the menstrual cycle and lower than those in the luteal phase [49]. An attenuating action of estradiol on stress-induced hypothalamopituitary adrenal axis activity in women is similar to the effects of estrogen on the autonomic nervous system but contrasts with the situation in rodents, where estradiol generally has facilitatory actions (see above). There has been considerably less research on the role of the other principal ovarian steroid, progesterone, in influencing the hypothalamo-pituitary adrenal axis. In rodents, progesterone has been reported to suppress the hypothalamo-pituitary adrenal axis response to stress [107-109] and there is now direct evidence to suggest that this is also the case in men, although there may be divergent effects on the sympatho-adrenal medullary system [110]. Administration of progesterone to men attenuated cortisol responses to the Trier Social Stress Test and reduced negative mood and alertness after stress but increased plasma norepinephrine and systolic blood pressure [110]. A similar approach has not been undertaken in women although in a study that utilised positive emotion-arousing there was a positive correlation between salivary progesterone and cortisol in men and in women taking hormonal contraceptives but not in women undergoing natural menstrual cycles, prompting the authors to suggest that progesterone may play a role in down-regulating the stress response [111]. Testosterone may also have a down regulating effect on the stress systems in humans because administration of testosterone to women resulted in decreased stress responsiveness [112-114], which is similar to the case in rodents (see above). While a role for testosterone in regulating the activity of the hypothalamo-pituitary adrenal axis was suggested [112], the collective work

regions [76-80]. For example, it has been demonstrated that estradiol benzoate treatment of ovariectomized rats increased extracellular levels of norepinephrine and dopamine in the pre-optic area of the hypothalamus during the dark phase [81], although this is not considered the predominant locus in regulating the sympatho-adrenal medullary system. Furthermore, sexually dimorphic responses in activation of the locus coeruleusnorepinephrine system have been shown in rats [82] although it is possible that this is more important to regulation of the hypothalamo-pituitary adrenal axis than the sympathoadrenal medullary system. An *in vitro* study showed that estradiol-17β stimulated catecholamine synthesis from adrenal medullary cells, which is direct evidence that this steroid is capable of influencing the sympatho-adrenal medullary system [83]. In human females, one study found that sympathetic nervous activity in response to mental stress was similar between the early follicular phase and mid-luteal phase but the recovery was prolonged during the mid-luteal phase [84]. A number of other investigations in women have reported that estrogen attenuates sympathetic activity in response to stress although this has not been found in all studies (for review see [49]). An attenuating effect on sympathetic activity in response to mental arithmetic was also demonstrated in young men treated with estradiol [85]. Nonetheless, these were not extended to the complete sympathoadrenal medullary system and little is known about the effects of progesterone and testosterone on this stress system.

The body of work in rodents and humans has generally suggested that females have higher basal glucocorticoid levels than males [86, 87]. In rodents, females also have higher stressinduced ACTH and glucocorticoid responses [88-90] than males and this is due, at least in part, to a stimulatory effect of estradiol on the hypothalamo-pituitary adrenal axis [91-93]. One possible mechanism for this may involve enhanced CRH expression because there are estrogen-responsive elements on the 5' regulatory region of the CRH gene [93]. Estradiol has also been shown to act directly on neurons within the paraventricular nucleus via estrogen receptor α [94]. Given this effect of estradiol it follows that reproductive state will affect the hypothalamo-pituitary adrenal axis and variations in activity occur across the estrous cycle of the rat [95, 96] and menstrual cycle of the monkey [97]. In contrast to estrogens, in rodents androgens are considered to inhibit the activity of the hypothalamo-pituitary adrenal axis [88, 92, 94, 98], possibly via a mechanism that involves direct actions of testosterone on neurons within the paraventricular nucleus via estrogen receptor β (for extensive review see [94]). It has been proposed that testosterone is converted to an androgenic metabolite, 5αandrostane-3β,17β-diol, that binds estrogen receptor β to regulate oxytocinergic neurons in the paraventricular nucleus [94]. Despite these opposing actions of estrogens and androgens on the activity of the hypothalamo-pituitary adrenal axis in rodents, the roles of sex steroids in influencing stress responses are less clear-cut in other species, and it is unknown if similar mechanism of action exist in non-rodent species.

We have not directly addressed the effects of steroids on the sympatho-adrenal medullary system in sheep but have considered sex differences and the importance of the gonads when it comes to the hypothalamo-pituitary adrenal axis. In adult sheep we identified a range of differences between males and females in the hypothalamo-pituitary adrenal axis and some, but not all, of these differences depended on gonadal factors [99]. Of significance in this study was an enhanced adrenocortical response to ACTH in females compared to males, and this occurred irrespective of the presence or absence of gonads. Males had higher AVP in the median eminence than females and gonadectomy increased this in both sexes [99].

regions [76-80]. For example, it has been demonstrated that estradiol benzoate treatment of ovariectomized rats increased extracellular levels of norepinephrine and dopamine in the pre-optic area of the hypothalamus during the dark phase [81], although this is not considered the predominant locus in regulating the sympatho-adrenal medullary system. Furthermore, sexually dimorphic responses in activation of the locus coeruleusnorepinephrine system have been shown in rats [82] although it is possible that this is more important to regulation of the hypothalamo-pituitary adrenal axis than the sympathoadrenal medullary system. An *in vitro* study showed that estradiol-17β stimulated catecholamine synthesis from adrenal medullary cells, which is direct evidence that this steroid is capable of influencing the sympatho-adrenal medullary system [83]. In human females, one study found that sympathetic nervous activity in response to mental stress was similar between the early follicular phase and mid-luteal phase but the recovery was prolonged during the mid-luteal phase [84]. A number of other investigations in women have reported that estrogen attenuates sympathetic activity in response to stress although this has not been found in all studies (for review see [49]). An attenuating effect on sympathetic activity in response to mental arithmetic was also demonstrated in young men treated with estradiol [85]. Nonetheless, these were not extended to the complete sympathoadrenal medullary system and little is known about the effects of progesterone and

The body of work in rodents and humans has generally suggested that females have higher basal glucocorticoid levels than males [86, 87]. In rodents, females also have higher stressinduced ACTH and glucocorticoid responses [88-90] than males and this is due, at least in part, to a stimulatory effect of estradiol on the hypothalamo-pituitary adrenal axis [91-93]. One possible mechanism for this may involve enhanced CRH expression because there are estrogen-responsive elements on the 5' regulatory region of the CRH gene [93]. Estradiol has also been shown to act directly on neurons within the paraventricular nucleus via estrogen receptor α [94]. Given this effect of estradiol it follows that reproductive state will affect the hypothalamo-pituitary adrenal axis and variations in activity occur across the estrous cycle of the rat [95, 96] and menstrual cycle of the monkey [97]. In contrast to estrogens, in rodents androgens are considered to inhibit the activity of the hypothalamo-pituitary adrenal axis [88, 92, 94, 98], possibly via a mechanism that involves direct actions of testosterone on neurons within the paraventricular nucleus via estrogen receptor β (for extensive review see [94]). It has been proposed that testosterone is converted to an androgenic metabolite, 5αandrostane-3β,17β-diol, that binds estrogen receptor β to regulate oxytocinergic neurons in the paraventricular nucleus [94]. Despite these opposing actions of estrogens and androgens on the activity of the hypothalamo-pituitary adrenal axis in rodents, the roles of sex steroids in influencing stress responses are less clear-cut in other species, and it is unknown if similar

We have not directly addressed the effects of steroids on the sympatho-adrenal medullary system in sheep but have considered sex differences and the importance of the gonads when it comes to the hypothalamo-pituitary adrenal axis. In adult sheep we identified a range of differences between males and females in the hypothalamo-pituitary adrenal axis and some, but not all, of these differences depended on gonadal factors [99]. Of significance in this study was an enhanced adrenocortical response to ACTH in females compared to males, and this occurred irrespective of the presence or absence of gonads. Males had higher AVP in the median eminence than females and gonadectomy increased this in both sexes [99].

testosterone on this stress system.

mechanism of action exist in non-rodent species.

Gonadectomy also elevated median eminence content of CRH but there was no difference between sexes. There was no effect of *in vitro* ACTH secretion in response to treatment with AVP, CRH and the two in combination [99]. Our research in prepubertal sheep has also shown that some sex differences in the activity of the hypothalamo-pituitary adrenal axis endure in the absence of the sex steroids [64]. Consistent with these findings, we also showed that sex and gonadal status affect the distribution of CRH, AVP and enkephalin in the paraventricular nucleus of sheep, providing a neuroanatomical basis for sex differences in the central regulation of the hypothalamo-pituitary adrenal axis [66]. Others have shown that steroids and reproductive state can affect the activity of the hypothalamo-pituitary adrenal axis in female sheep [100], but there were no comparisons with males. In a series of studies in ewes it was shown that progesterone inhibits ACTH stimulation of cortisol [101, 102, 103] and stress, induced secretion of cortisol estrogens and androgens influence hypothalamic concentrations of CRH and AVP [104], and that ovariectomy affects hypothalamo-pituitary adrenal axis responses to stress [105, 106]. While it is clear that gonadal factors, such as the sex steroids, and reproductive state, influence the activity of the hypothalamo-pituitary adrenal axis in sheep, there is a need for more extensive research to establish the precise roles of steroids and reproductive state, including with regard to different stressors.

In women the stage of the reproductive cycle has been shown to affect the hypothalamopituitary adrenal axis responses to stress (for an extensive review see [49]). It has been consistently reported that cortisol responses to psychosocial stress were lower in women in the follicular phase of the menstrual cycle compared with women in the luteal phase of the menstrual cycle [49]. This suggests that there is attenuation of the hypothalamo-pituitary adrenal axis in response to psychosocial stress in an estrogenic environment (i.e. the follicular phase), and this is supported by studies showing that women taking a synthetic estrogen as an oral contraceptive had similar cortisol responses to psychosocial stress compared to women in the follicular phase of the menstrual cycle and lower than those in the luteal phase [49]. An attenuating action of estradiol on stress-induced hypothalamopituitary adrenal axis activity in women is similar to the effects of estrogen on the autonomic nervous system but contrasts with the situation in rodents, where estradiol generally has facilitatory actions (see above). There has been considerably less research on the role of the other principal ovarian steroid, progesterone, in influencing the hypothalamo-pituitary adrenal axis. In rodents, progesterone has been reported to suppress the hypothalamo-pituitary adrenal axis response to stress [107-109] and there is now direct evidence to suggest that this is also the case in men, although there may be divergent effects on the sympatho-adrenal medullary system [110]. Administration of progesterone to men attenuated cortisol responses to the Trier Social Stress Test and reduced negative mood and alertness after stress but increased plasma norepinephrine and systolic blood pressure [110]. A similar approach has not been undertaken in women although in a study that utilised positive emotion-arousing there was a positive correlation between salivary progesterone and cortisol in men and in women taking hormonal contraceptives but not in women undergoing natural menstrual cycles, prompting the authors to suggest that progesterone may play a role in down-regulating the stress response [111]. Testosterone may also have a down regulating effect on the stress systems in humans because administration of testosterone to women resulted in decreased stress responsiveness [112-114], which is similar to the case in rodents (see above). While a role for testosterone in regulating the activity of the hypothalamo-pituitary adrenal axis was suggested [112], the collective work

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

and females in different physiological states.

**8. Acknowledgements** 

p. 115-130.

224: p. 452-459.

**9. References** 

Medullary System and Hypothalamo-Pituitary Adrenal Axis Responses to Stress 129

due to gonadal factors, such as the sex steroids, but others are not. There also appear to be differences between species in the impact of sex steroids on the stress systems. For example, whereas estrogens appear to facilitate and androgens abrogate stress responses in rodents, the actions of estrogens on stress responses in humans may be the opposite, at least in some cases, and the effects of androgens have received insufficient research in non-rodent species to draw definitive conclusions. Nevertheless, since there are effects of sex steroids on stress responsiveness, there are differences in stress responsiveness at different stages of the female reproductive cycle. Furthermore, during lactation, stress responses are generally attenuated,

This review has highlighted substantial gaps in knowledge that are required to fully appreciate the field of stress physiology. These include understanding mechanisms of stress responses to different types of stressors, between sexes and in different physiological states. They also include the mechanisms by which different stressors impact the body of males

We acknowledge Deakin University and Monash University for their support. We are grateful to the Australian Research Council and National Health and Medical Research Council of Australia for funding the research that generated the data presented in this

[1] Tilbrook, A.J. and I.J. Clarke, *Neuroendocrine mechanisms of innate states of attenuated* 

[2] Tilbrook, A.J., *Neuropeptides, Stress-Related*, in *Encyclopedia of stress*, G. Fink, Editor. 2007,

[3] Johnson, E.O., et al., *Mechanisms of stress: a dynamic overview of hormonal and behavioral* 

[5] Selye, H., *The general adaptation syndrome and the diseases of adaption.* Journal of Clinical

[6] Goldstein, D.S. and A. Grossman, *Stress-induced activation of the sympathetic nervous system*, in *Neuroendocrinology of Stress*. 1987, Bailliere Tindall: London. p. 253-278. [7] Axelrod, J. and T.D. Reisine, *Stress hormones: their interaction and regulation.* Science, 1984.

[8] Stackpole, C.A., et al., Seasonal differences in the effect of isolation and restraint stress

[10] Vale, W., et al., *Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and β-endorphin.* Science, 1981. 213: p. 1394-1397.

on the luteinizing hormone response to gonadotropin-releasing hormone in hypothalamopituitary disconnected, gonadectomized rams and ewes. Biology of

*responsiveness of the hypothalamo-pituitary adrenal axis to stress.* Frontiers in

*homeostasis. [Review] [218 refs].* Neuroscience & Biobehavioral. Reviews., 1992. 16(2):

unless the stressor threatens the well-being of the mother and, in turn, the offspring.

chapter. We also thank Sara Drew for proof reading this manuscript.

Neuroendocrinology, 2006. 27(3): p. 285-307.

[4] Cannon, W., *The Wisdom of the Body*. 1932, New York: Norton.

Reproduction, 2003. 69(4): p. 1158-1164.

Endocrinology and Metabolism, 1946. 6: p. 117-230.

[9] Harris, G.W., Neural control of the pituitary gland. 1955, London: Arnold.

Academic Press: Oxford. p. 903-908.

of these authors (e.g. [112-114]) also implicates a regulatory role for testosterone on the autonomic nervous system and, possibly, the sympatho-adrenal medullary system. Further research is necessary to confirm this and the research needs to be extended to males.

Lactation is a physiological state that can have profound effects on both the basal and stressinduced activity of the hypothalamo-pituitary adrenal axis. In many species, including humans, ungulates and rodents, it has been consistently found that lactating females show attenuated neuroendocrine responses to stress (for reviews see [115-117]) and anxietyrelated behaviours (for reviews see [116, 118, 119]). The alterations in the hypothalamopituitary adrenal axis that result in reduced responses to stress begin to emerge in late pregnancy and occur in a continuum throughout lactation (for reviews see [116, 118, 120, 121]). Although the mechanisms for attenuated hypothalamo-pituitary adrenal axis responses to stress during lactation are not fully known, it appears that they include reduced synthesis and secretion of CRH and AVP due to enhanced negative feedback by glucocorticoids and/or reduced noradrenergic stimulatory input from the brain stem, reduced pituitary responsiveness to CRH and AVP and, possibly, inhibition by oxytocin and prolactin [1]. We have shown in sheep that the greatest attenuation of the hypothalamopituitary adrenal axis is achieved when the lactating mother is suckled [122] and this is likely to also be the case for humans when breastfeeding [1]. Despite the many published reports of attenuated stress responses in lactating females this has not always been the case and the nature of the stressor seems to be important. It has been shown in both sheep [123] and humans [87] that the there is activation of the hypothalamo-pituitary adrenal axis in response to a stressor that may threaten the welfare of the infant by virtue of harming the mother. This makes perfect sense given that the mother would require a stress response in order to dispose of the threat posed to herself and her offspring. This underscores the importance of being able to mount stress responses so that homeostasis can be restored (Section 1).

#### **7. Conclusions**

Although there are various ways to define stress there is generally acceptance that stress responses occur in response to noxious stimuli, whether perceived or real, commonly called stressors. A range of physiological systems are activated with the two of the most prominent being the sympatho-adrenal medullary system and the hypothalamo-pituitary adrenal axis. The catecholamines and glucocorticoids, released by each system respectively, have farreaching effects within the body to re-establish the homeostasis that was disrupted by stressors. Such stress responses are vital for a healthy life. Nevertheless, when the stress systems are frequently or continually activated, the on-going action of the catecholamines and glucocorticoids can be destructive and lead to pathological conditions. An inability to mount an appropriate stress response may also result in illness. Therefore, it follows that understanding stress responses, and the factors that affect stress responses, is paramount to develop strategies and treatments to avoid or cure stress-induced disorders and pathologies. These factors include sex, sex steroids and physiological state, particularly reproductive state. There are differences between males and females in various illnesses and pathological states and many of these are those induced or exacerbated by frequent or chronic stress. Males and females respond differently to some stressors and not others. The implications for health and survival of these different stress responses are unknown and research is required to determine this. At least some of the mechanisms for sex differences in stress responses are due to gonadal factors, such as the sex steroids, but others are not. There also appear to be differences between species in the impact of sex steroids on the stress systems. For example, whereas estrogens appear to facilitate and androgens abrogate stress responses in rodents, the actions of estrogens on stress responses in humans may be the opposite, at least in some cases, and the effects of androgens have received insufficient research in non-rodent species to draw definitive conclusions. Nevertheless, since there are effects of sex steroids on stress responsiveness, there are differences in stress responsiveness at different stages of the female reproductive cycle. Furthermore, during lactation, stress responses are generally attenuated, unless the stressor threatens the well-being of the mother and, in turn, the offspring.

This review has highlighted substantial gaps in knowledge that are required to fully appreciate the field of stress physiology. These include understanding mechanisms of stress responses to different types of stressors, between sexes and in different physiological states. They also include the mechanisms by which different stressors impact the body of males and females in different physiological states.
