G-protein coupled estrogen receptor, estrogen receptor α, and progesterone receptor immunohistochemistry in the hypothalamus of aging female rhesus macaques given long-term estradiol treatment.

Reproductive aging in females is highly divergent among mammalian species. Menopause is limited to those few species that menstruate (humans, great apes, and some non-human primates); it is a natural transition to reproductive senescence associated with decreased levels of the sex steroid hormones estradiol (E2) and progesterone (P4) (Trevoux et al., ‘86; Burger et al., 2002). Estrogens and progestins are not only critical for reproduction, but also play significant roles in the normal functioning of brain networks, cardiovascular systems, bone maintenance, and many others (Baulieu and Robel, ‘90; Inoue, 2002; McEwen, 2002). In women, the menopausal decline in circulating hormones is often accompanied by symptoms that have a dramatic negative impact on quality of life such as mood alterations, sleep disruptions, increased risk of osteoporosis and more. There are many available treatments for mitigation of menopausal symptoms, the most common being hormone replacement therapy (HRT) containing estrogens, or estrogens in combination with progestins. The risks versus benefits of health outcomes are highly controversial (Herrinton and Weiss, ‘93; Fitzpatrick et al., 2000; Rossouw et al., 2002; Canonico et al., 2008; Talboom et al., 2008; Prentice et al., 2009; Terauchi et al., 2012; Manson et al., 2013), with differential results due in part to variations in hormone formulations and timing/duration of hormone treatment relative to the menopausal transition. Non-human primates undergo many similar neurobiological (functional and cellular) and physical (e.g., osteoporosis, metabolic) alterations with menopause as in women (Hao et al., 2003, 2007; Rapp et al., 2003; Maffucci and Gore, 2006). Furthermore, mammalian species that do not menstruate may also undergo a loss of reproductive capacity with aging, often very differently from primates due to those species’ unique reproductive properties such as strong seasonal breeding period, estrous cycles or induced ovulation (as opposed to spontaneous ovulation and reproductive cycles), and other reproductive traits (Maffucci and Gore, 2006; Kermath and Gore, 2012). Although there may be variability, a conserved property across species is that reproductive senescence involves the three levels of the hypothalamic-pituitary-gonadal (HPG) axis. Declines in hypothalamic function precede ovarian failure in rodents and primates, although the ovary may play a more primary role in women (Wise, ‘84; Richardson et al., ‘87; Gougeon et al., ‘94; Gore et al., 2000; Gill et al., 2002a,b; Weiss et al., 2004; Downs and Wise, 2009). While age-related changes in positive and negative feedback on gonadotropin-releasing hormone (GnRH) and gonadotropin release clearly occur in rodents, the evidence is less clear for both non-human and human primates (Van Look et al., ‘77; Wise and Ratner, ‘80; Gore et al., 2000; Tsai et al., 2004; Hall, 2007; Downs and Wise, 2009; Rance, 2009; Shaw et al., 2011). The neurons that synthesize GnRH are modulated by ovarian hormonal feedback both directly and indirectly via steroid hormone receptors, including G protein-coupled estrogen receptor (GPER), estrogen receptor α (ERα), and progesterone receptor (PR), among others (Van Look et al., ‘77; Liu and Yen, ‘83; Sullivan et al., ‘95; Terasawa, ‘95; Skinner et al., ‘98; Wilson et al., 2002; Dorling et al., 2003; Petersen et al., 2003; Glidewell-Kenney et al., 2007). An important research gap is whether, and how, hormone feedback on the hypothalamus may change with aging, and on which cells these effects are mediated. The mechanism for these changes is also unclear, although it may involve age-related change in expression or function of the steroid hormone receptors that mediate steroid hormone effects [reviewed in Chakraborty and Gore (2004)]. In the current study, we addressed this question in female monkeys as a translational model for the neurobiology of menopause in women (Gilardi et al., ‘97; Kaplan, 2004). Rhesus monkeys have 28-day menstrual cycles and undergo natural reproductive senescent changes that mirror the human menopausal transition, albeit much later in life (Krey et al., ‘75; Gilardi et al., ‘97; Archer, 2004; Gore et al., 2004). We focused our work on two sub-regions of the hypothalamus involved in HPG function in primates, the arcuate nucleus (ARC) and the periventricular region (PERI), which are integral to reproduction, growth, thermoregulation and metabolism (Wiegand and Terasawa, ‘82; Hofman, ‘97; Downs and Wise, 2009; Castellanoa et al., 2010; Mittelman-Smith et al., 2012). These hypothalamic areas also express high levels of steroid hormone receptors and are important targets of E2 feedback in the regulation of the HPG axis (Bethea et al., ‘96; Skinner et al., ‘98; Blurton-Jones et al., ‘99; Mills et al., 2002; Petersen et al., 2003; Rapp et al., 2003; Tsai et al., 2004; Michael et al., 2005). To determine whether there are age-related changes in steroid hormone receptors, and altered responses of these receptors to E2 feedback, we quantified the density and percentage of cells that express GPER, ERα, and PR in the ARC and PERI of young and aged macaques that were ovariectomized (OVX) and given E2 or vehicle treatment for 2 years. Because relatively little is known about the distribution of GPER neurons in the adult brain, we also mapped their localization across the hypothalamus.