Chapter 2 Estrogen modulates oxytocin gene expression in regions of the rat supraoptic and paraventricular nuclei that contain estrogen receptor-β

Abstract

Oxytocin is an important modulator of female reproductive functions including parturition, lactation and maternal behavior, while vasopressin regulates water balance and acts as a neurotransmitter. For decades, it has been suggested that estrogen regulates the production and/or release of oxytocin and vasopressin in the rodent brain. Although several studies demonstrated that estrogen can modulate vasopressin mRNA levels in regions known to contain estrogen receptor (ER), such as the bed nucleus of the stria terminalis and medial amygdala, data from the paraventricular and supraoptic nuclei were inconclusive. Since early immunohistochemical and in situ hybridization studies revealed few, if any, ER containing cells in these hypothalamic nuclei, it was thought that oxytocin and vasopressin were not directly regulated by estrogen. The discovery of a second ER (ER-beta) in the late 1990s suggested that estrogen could act in many brain regions heretofore not considered targets for estrogen action. Initial in situ hybridization studies revealed a wide distribution of ER-beta mRNA in the rat brain including neurons of the supraoptic nucleus and the parvocellular and magnocellular divisions of the paraventricular nucleus. Subsequent double-label in situ hybridization/immunocytochemistry studies showed that ER-beta mRNA was present in oxytocin and vasopressin neurons, with the degree of colocalization being both neuropeptide and region specific. In an attempt to demonstrate that ER-beta mRNA was translated into a biologically active protein, a series of in vivo binding studies were conducted in rats with 125I-estrogen. These data revealed the presence of nuclear estrogen binding sites in neurons of the magnocellular system indicating that ER-beta mRNA was translated into protein. Concurrent studies in mice found that the distribution of ER-beta mRNA and 125I-estrogen binding was similar to rats, although there were some notable differences. For example, ER-beta mRNA and binding were not detected in the mouse supraoptic nucleus and although ER-beta was the principle ER in the paraventricular nucleus, ER-alpha was also present. The prevalence of ERs in the mouse paraventricular nucleus was further investigated using ER-alpha and ER-beta knockout mice for in vivo binding studies with 125I-estrogen. The results of these studies showed that ER-beta was the predominant ER in the paraventricular nucleus and confirmed the presence of ER-beta in other brain regions. Moreover, our group recently generated and characterized several polyclonal antisera raised against the C-terminus of ER-beta. Through the use of these antisera, we have confirmed the presence of ER-beta in the rat paraventricular and supraoptic nuclei and shown that ER-beta is colocalized, in part, with oxytocin and vasopressin. To assess the ability of estrogen to modulate the expression of oxytocin mRNA, ovariectomized rats were treated with vehicle or estradiol and the brains processed for in situ hybridization. The results of these studies revealed that estradiol down-regulated oxytocin mRNA in the rat paraventricular nucleus within 6 h of treatment. Together these data and the observation that some of the oxytocin and vasopressin neurons contain ER-beta suggest that estrogen, acting through ER-beta, may directly regulate oxytocin gene expression. However, since the paraventricular nucleus has many subdivisions with different projections and the degree of colocalization of ER-beta with oxytocin/vasopressin varies among subdivisions, the effects of estrogen treatment on gene expression requires further study to ascertain the role of estrogen action in this neuronal systems.