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