Elsevier

Psychoneuroendocrinology

Volume 34, Issue 9, October 2009, Pages 1314-1328
Psychoneuroendocrinology

Role of testosterone in mediating prenatal ethanol effects on hypothalamic–pituitary–adrenal activity in male rats

https://doi.org/10.1016/j.psyneuen.2009.04.001Get rights and content

Summary

Prenatal ethanol (E) exposure programs the fetal hypothalamic–pituitary–adrenal (HPA) and –gonadal (HPG) axes such that E rats show HPA hyperresponsiveness to stressors and altered HPG and reproductive function in adulthood. Importantly, prenatal ethanol may differentially alter stress responsiveness in adult male and female offspring compared to their control counterparts. To test the hypothesis that alterations in HPA activity in E males are mediated, at least in part, by ethanol-induced changes in the capacity of testosterone to regulate HPA activity, we explored dose-related effects of testosterone on HPA and HPG function in adult male offspring from prenatal E, pair-fed (PF) and ad libitum-fed control (C) dams. Our data suggest that E males show changes in both HPA and HPG regulation, as well as altered sensitivity to the inhibitory effects of testosterone. While gonadectomy (GDX) reduced weight gain in all animals, low testosterone replacement restored body weights in PF and C but not E males. Further, sensitivity of the thymus and adrenal to circulating testosterone was reduced in E rats. In addition, stress-induced corticosterone (CORT) levels were increased in PF and C but not E males following GDX, and while low dose testosterone replacement restored CORT levels for PF and C, high testosterone levels were needed to normalize CORT levels for E males. A negative correlation between pre-stress testosterone and post-stress CORT levels in C but not in E and PF males further supports the finding of reduced sensitivity to testosterone. Importantly, testosterone appeared to have reduced effects on central corticotrophin releasing hormone (CRH) pathways in E, but greater effects on central arginine vasopressin (AVP) pathways in E and/or PF compared to C males. Testosterone also had less of an inhibitory effect on stress-induced luteinizing hormone increases in E than in PF and C males following GDX. In addition, androgen receptor mRNA levels in the medial preoptic nucleus and the principal nucleus of posterior bed nucleus of the stria terminalis were lower in E and PF compared to C males under intact conditions.

Together, these data support our previous work suggesting altered sensitivity to testosterone in E males. Furthermore, differential effects of testosterone on the complex balance between central CRH and central AVP pathways may play a role in the HPA alterations observed. That some findings were similar in E and PF males suggest that nutritional effects of diet may have played a role in mediating at least some of the changes seen in E animals.

Introduction

Fetal alcohol spectrum disorder (FASD) refers to the wide range of abnormalities or deficits that result from prental alcohol exposure (Hoyme et al., 2005). The effects range from physical and physiological abnormalities to altered cognitive and behavioral function, compromising an individual's ability to adapt to his/her environment. Work in our lab has focused on the effects of prenatal ethanol exposure on responses to stress and hypothalamic–pituitary–adrenal (HPA) regulation in adult offspring. Both clinical and animal studies have shown that ethanol exposure in utero results in HPA hyperactivity (Jacobson and Jacobson, 1999, Lee et al., 2000, Ramsay et al., 1996, Taylor et al., 1984, Weinberg, 1989). Importantly, however, while HPA hyperresponsiveness is a robust phenomenon, differential effects of prenatal ethanol exposure may be observed in male and female offspring, depending on the nature and intensity of the stressor, and the time course and hormonal endpoint examined (Lee and Rivier, 1996, Taylor et al., 1983, Taylor et al., 1988, Weinberg, 1988, Weinberg, 1992, Weinberg et al., 2008). These findings raise the possibility that ethanol-induced alterations in the gonadal hormones and/or in HPA–hypothalamic–pituitary–gonadal (HPG) interactions may play a role in mediating prenatal ethanol effects on HPA activity in adulthood.

Effects of the gonadal hormones on HPA function have been demonstrated at different levels of the axis. In general, estradiol activates, and androgens inhibit HPA activity. For example, androgens inhibit corticotrophin releasing hormone (CRH) expression (Bingaman et al., 1994), and gonadectomy (GDX) of adult male rats increases both adrenocorticotropin (ACTH) and CORT responses to physical and psychological stressors (Handa et al., 1994), an effect reversed by replacement with testosterone or the non-aromatizable androgen, 5α-dihydrotestosteone (5α-DHT) (Handa et al., 1994; Viau et al., 2003). GDX rats also show greater stress-induced Fos expression and higher arginine vasopressin (AVP) hnRNA levels than intact males, both of which are negatively correlated with plasma testosterone levels (Viau et al., 2003).

HPA responses are driven by neurons located in the medial parvocellular subdivision of the paraventricular nucleus (mpd PVN) of the hypothalamus. The mpd PVN contains both CRH- and AVP-expressing neurosecretory neurons, and is the final pathway that integrates multiple excitatory and inhibitory inputs from other brain areas regulating the HPA axis. Mapping studies have demonstrated that androgen receptors (ARs) are not localized in mpd PVN, but restricted to the parvocellular ventral division (mpv PVN) and the periventricular and dorsal parvocellular areas of the PVN (Bingham et al., 2006, Shughrue et al., 1997, Simerly et al., 1990, Zhou et al., 1994), suggesting that androgens act upstream from the PVN to regulate HPA output. Candidate brain areas for androgenic effects are the medial preoptic area (MPOA) [ARs are densest in the medial preoptic nucleus (MPN) of the MPOA], bed nucleus of the stria terminalis (BNST), amygdala, and hippocampus, regions that all contain high densities of ARs (Bingham et al., 2006, Kerr et al., 1995, Shughrue et al., 1997, Simerly et al., 1990, Williamson and Viau, 2007, Zhou et al., 1994). Furthermore, the anterior and posterior divisions of the BNST contain CRH- and AVP-projecting cells that terminate in the PVN (Champagne et al., 1998, Moga and Saper, 1994), and relay information to the PVN from the central and medial nuclei of the amygdala (Dong et al., 2001a, Dong et al., 2001b, Prewitt and Herman, 1998). The amygdala is activated during stress primarily by ascending catecholaminergic neurons in the brainstem or by emotional stressors, and the activation of these neurons leads to anxiety, fear, and stress system activation (Davis, 1992).

In a previous study, we found that intact E rats show increased ACTH but blunted testosterone and luteinizing hormone (LH) responses to restraint stress, and no stress-induced elevation in AVP mRNA levels compared to PF and/or control rats. Importantly, GDX significantly increased ACTH responses to stress in control but not E and PF males, eliminated differences among groups in plasma ACTH and AVP mRNA levels, and altered LH and gonadotropin-releasing hormone (GnRH) responses in E males. These findings indicate that central regulation of both the HPA and HPG axes are altered by prenatal ethanol exposure, with normal testicular influences on HPA function markedly reduced in E males (Lan et al., 2006).

To explore more directly the effects of testosterone on HPA regulation and responsiveness, the present study examined HPA and HPG activity in male rats under intact (sham GDX) conditions, and following GDX, with or without testosterone replacement at low or high concentrations. Our experimental questions were: (1) Do E males differ from controls in adrenal and gonadal hormone levels under intact conditions, following GDX and following GDX with low or high testosterone replacement? (2) Do differences between E and control animals with different circulating testosterone levels occur under both basal and stress conditions? (3) Do central measures of HPA and HPG activity differ in E compared to control males under different circulating testosterone levels? and (4) Is activity of central testosterone-sensitive pathways that regulate CRH and AVP neurosecretory neurons altered in E compared to controls males? We tested the hypothesis that the differential alterations in HPA activity observed in E males compared to their control counterparts are mediated, at least in part, by ethanol-induced changes in the capacity of testosterone to regulate HPA activity.

Section snippets

Animals and breeding

Male (275–300 g, n = 18) and female (230–275 g, n = 46) Sprague–Dawley rats were obtained from Charles River Laboratories (St. Constant, PQ, Canada). Rats were group-housed by sex until breeding and maintained on a 12:12 h light/dark cycle (lights on at 06:00 h), with controlled temperature (21–22 °C), and ad libitum access to standard lab chow and water. Animals were bred 1–2 weeks following arrival. All animal use and care procedures were in accordance with the National Institutes of Health Guidelines

Adult body and organ weights

Body weights. Analysis of adult body weights prior to surgery indicated a significant main effect of prenatal group (F(2,154) = 4.0; P < 0.05); E and PF had lower weights than C males (P < 0.05). Analysis of weight gain over the 2 weeks between surgery and testing (Fig. 1A) indicated significant effects of surgical condition (F(3,154) = 12.839; P < 0.001). Overall, INT males gained the most, and GDX and GDX-H males gained the least, weight (INT > GDX = GDX-H, Ps < 0.05; INT > GDX-L, P < 0.07; GDX-L > GDX, P < 0.05).

Discussion

The present findings support and extend those of our previous work (Lan et al., 2006), providing strong evidence that regulation of both the HPA and HPG axes is altered by prenatal ethanol exposure, and that E males show altered sensitivity to testosterone. Importantly, examination of central CRH and AVP expression profiles demonstrates a complex balance of effects; that is, testosterone appears to have a reduced effect on central CRH pathways, but an increased effect on central AVP pathways in

Summary and conclusions

In summary, examination of dose-related effects of testosterone unmasked alterations in HPA activity in E males that relate specifically to testosterone status, as well as central changes in both HPA and HPG regulation that are specific to prenatal ethanol exposure. We propose that although basal HPA hormone levels and CRH/AVP mRNA expression in the mpd PVN appear unchanged compared to those in control males, prenatal ethanol exposure significantly alters upstream CRH and AVP pathways that

Role of funding sources

This research was supported by NIH/NIAAA AA007789 to JW and VV, grants from the Canadian Institute for Advanced Research and the UBC Human Early Learning Partnership to JW, an NSERC Canada Graduate Scholarship to NL, Fellowships from IMPART (CIHR Strategic Training Initiative in Health Research) and the Michael Smith Foundation for Health Research to KH. The funding agencies have no further role in study design, in the collection, analysis and interpretation of data; in the writing of the

Conflict of interest

None declared.

Acknowledgements

A portion of these data has been presented orally at the International Society for Developmental Psychobiology (ISDP) 39th Annual Meeting, 2006, Atlanta, supported by Sandra G. Wiener Award from ISDP to NL. We thank Stephanie Westendorp, Paxton Pach, Teri Herbert, Yun Han and Wayne K. Yu for their technical assistance on the experiments.

References (71)

  • R.J. Handa et al.

    Androgen regulation of adrenocorticotropin and corticosterone secretion in the male rat following novelty and foot shock stressors

    Physiol. Behav.

    (1994)
  • P. Kehoe et al.

    Opioid-dependent behaviors in infant rats: effects of prenatal exposure to ethanol

    Pharmacol. Biochem. Behav.

    (1991)
  • O. Kofman

    The role of prenatal stress in the etiology of developmental behavioural disorders

    Neurosci. Biobehav. Rev.

    (2002)
  • S. Lee et al.

    Gender differences in the effect of prenatal alcohol exposure on the hypothalamic–pituitary–adrenal axis response to immune signals

    Psychoneuroendocrinology

    (1996)
  • S. Lee et al.

    Increased activity of the hypothalamic–pituitary–adrenal axis of rats exposed to alcohol in utero: role of altered pituitary and hypothalamic function

    Mol. Cell. Neurosci.

    (2000)
  • S.G. Matthews

    Early programming of the hypothalamo–pituitary–adrenal axis

    Trends Endocrinol. Metab.

    (2002)
  • B.B. Turner et al.

    Sexual dimorphism of glucocorticoid binding in rat brain

    Brain Res.

    (1985)
  • C.V. Vorhees

    A fostering/crossfostering analysis of the effects of prenatal ethanol exposure in a liquid diet on offspring development and behavior in rats

    Neurotoxicol. Teratol.

    (1989)
  • G.N. Wade

    Sex hormone, regulatory behaviors, and body weight

  • J. Weinberg

    Prenatal ethanol effects: sex differences in offspring stress responsiveness

    Alcohol

    (1992)
  • M. Williamson et al.

    Central organization of androgen-sensitive pathways to the hypothalamic–pituitary–adrenal axis: implications for individual differences in responses to homeostatic threat and predisposition to disease

    Prog. Neuropsychopharmacol. Biol. Psychiatry

    (2005)
  • R.Y. Yukhananov et al.

    Estrogen alters proenkephalin RNAs in the paraventricular nucleus of the hypothalamus following stress

    Brain Res.

    (1997)
  • R.S. Ahima et al.

    Sexual dimorphism in regulation of type II corticosteroid receptor immunoreactivity in the rat hippocampus

    Endocrinology

    (1992)
  • S. Barron et al.

    Neonatal ethanol exposure but not neonatal cocaine selectively reduces specific isolation-induced vocalization waveforms in rats

    Behav. Genet.

    (2005)
  • E.W. Bingaman et al.

    Androgen inhibits the increases in hypothalamic corticotropin-releasing hormone (CRH) and CRH-immunoreactivity following gonadectomy

    Neuroendocrinology

    (1994)
  • B. Bingham et al.

    Androgen and estrogen receptor-beta distribution within spinal-projecting and neurosecretory neurons in the paraventricular nucleus of the male rat

    J. Comp. Neurol.

    (2006)
  • R.N. Cardinal et al.

    ANOVA for the Behavioural Sciences Researcher

    (2006)
  • D. Champagne et al.

    CRFergic innervation of the paraventricular nucleus of the rat hypothalamus: a tract-tracing study

    J. Neuroendocrinol.

    (1998)
  • M. Davis

    The role of the amygdala in fear and anxiety

    Annu. Rev. Neurosci.

    (1992)
  • E.R. De Kloet et al.

    Brain corticosteroid receptor balance in health and disease

    Endocr. Rev.

    (1998)
  • G.J. De Vries et al.

    Sex differences in the effects of testosterone and its metabolites on vasopressin messenger RNA levels in the bed nucleus of the stria terminalis of rats

    J. Neurosci.

    (1994)
  • H.W. Dong et al.

    Basic organization of projections from the oval and fusiform nuclei of the bed nuclei of the stria terminalis in adult rat brain

    J. Comp. Neurol.

    (2001)
  • F. Faul et al.

    G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences

    Behav. Res. Methods

    (2007)
  • R.T. Gentry et al.

    Androgenic control of food intake and body weight in male rats

    J. Comp. Physiol. Psychol.

    (1976)
  • M.M. Glavas et al.

    Effects of prenatal ethanol exposure on basal limbic–hypothalamic–pituitary–adrenal regulation: role of corticosterone

    Alcohol. Clin. Exp. Res.

    (2007)
  • Cited by (24)

    • Neurodevelopment in adolescents and adults with fetal alcohol spectrum disorders (FASD): A magnetic resonance region of interest analysis

      2020, Brain Research
      Citation Excerpt :

      Testosterone plays an important role in increasing neuronal density, size, and synaptogenesis (Goldstein et al., 2001), and in humans with prenatal alcohol exposure, testosterone levels are lower in both males and females (Carter et al., 2014). Furthermore, preclinical studies have shown that male rats with prenatal alcohol exposure are less sensitive to the neurophysiological effects of testosterone (Lan et al., 2009); it is possible that the effects of alcohol on the hormonal system are an underlying mechanism for the smaller region volumes observed in males with FASD (Chen et al., 2012). Regardless, our findings suggest that the relationship between prenatal alcohol exposure and brain structure is dependent on sex, making it an important factor to consider when examining the brain structure of individuals with FASD.

    • Sexual dimorphism of volume reduction but not cognitive deficit in fetal alcohol spectrum disorders: A combined diffusion tensor imaging, cortical thickness and brain volume study

      2017, NeuroImage: Clinical
      Citation Excerpt :

      However, animal models of prenatal alcohol exposure have revealed several sex-dependent neurophysiological and neurochemical sequelae, many of which appear to be more severe in males than females. Examples of PAE effects observed to be greater in males than females include reduced preoptic area of the hypothalamus (Barron et al., 1988), 40% reductions of long term potentiation (Sickmann et al., 2014), increases in dopamine D1R binding (Converse et al., 2014), reduced sensitivity to testosterone (Lan et al., 2009), as well as greater degrees of structural abnormality in the corpus callosum (Zimmerberg and Reuter, 1989). Prenatal alcohol exposure also appears to produce complex sexually dimorphic effects such as up-regulation of the hypothalamic-pituitary axis that depends on different concomitant environmental factors in males than females (Weinberg et al., 2008).

    • Evidence for an immune signature of prenatal alcohol exposure in female rats

      2016, Brain, Behavior, and Immunity
      Citation Excerpt :

      Finally, it will be important in future studies to determine whether similar changes in endocrine and immune function also occur in PAE male offspring. The finding that PAE and PF dams both weighed less than controls is not surprising, as we reduce the food ration of PF dams to match that of PAE dams, and supports previous data from our laboratory (Hellemans et al., 2008; Lan et al., 2009; Uban et al., 2010) and others (Abel, 1978; Thomas et al., 2000). Consistent with these findings were lower body and brain weights in PAE and PF compared to control pups at birth, with catch-up growth by P8.

    • Association of testosterone and BDNF serum levels with craving during alcohol withdrawal

      2016, Alcohol
      Citation Excerpt :

      Mehta et al. (2015) reported that risk taking and testosterone serum levels were positively associated in male and female probands when HPA activity was low, suggesting a link between impulsivity, testosterone release, and HPA activity. Partly closing the gap between alterations of testosterone levels, HPA activity, and alcohol consumption, prenatal exposure to ethanol was reported to reduce testosterone's effect on the HPA (Lan, Hellemans, Ellis, Viau, & Weinberg, 2009). Moreover, corticosterone replacement therapy was reported to result in increased alcohol consumption in adrenalectomized alcohol-preferring male rats, implicating a direct corticosterone effect on alcohol consumption in vulnerable populations (Fahlke & Eriksson, 2000).

    View all citing articles on Scopus
    View full text