Oestrogen Receptor b-Immunoreactive Neurones in the Ovine Hypothalamus: Distribution and Colocalisation with Gonadotropin-Releasing Hormone D. C. Skinner and L. Dufourny University of Wyoming, Department of Zoology and Physiology, Biological Science Building, Laramie, WY, USA. Key words: sex steroid receptors, antigen retrieval technique, sheep, GnRH, oestradiol. Abstract Oestrogen powerfully affects the secretion of gonadotropin-releasing hormone (GnRH) from the brain in all species investigated, including sheep. Until recently, it was hypothesised that such regulation occurs indi- rectly because few or no GnRH neurones were found to express oestrogen receptor (ER) a. The discovery of a second oestrogen receptor, ERb, and its subsequent localisation in numerous GnRH neurones in the rat, led to a reconsideration of this hypothesis. However, colocalisation of immunoreactive ERb protein in GnRH neurones has only been demonstrated in the rat, raising the possibility that such putative direct regulation of GnRH neurones by oestrogen may be peculiar to this species. We have previously shown that steroid receptors in the sheep brain are acutely sensitive to fixation and the full complement of immunoreactive cells can only be visualised after antigen retrieval. The aims of this study were therefore to map immu- nocytochemically the distribution of ERb neurones in the ewe brain, and to determine which proportion of GnRH neurones express ERb. Brain sections (20 lm) from four ewes killed in anestrus were subjected to high temperature antigen retrieval and immunocytochemistry. Numerous ERb-immunoreactive cells were located throughout the hypothalamus and, following dual-label immunocytochemistry, over 50% of the GnRH neurones were found to express immunoreactive ERb. The functional significance of these ERb- expressing GnRH neurones in the ovine brain remains to be determined. Oestrogens have long been known to regulate the activity of the gonadotropin-releasing hormone (GnRH)/luteinising hormone (LH) system throughout the reproductive cycle in many species, including sheep (1). However, earlier studies in numerous species suggested that GnRH neurones were not directly regulated by oestrogen because oestrogen receptors (ER) were not found in those neurones (2–7), and that interneurones were necessary for the transduction of oestrogen signals to the GnRH system. This idea was reinforced by studies demonstrating that micro-implants of oestrogen in the ventromedial nucleus, but not in the preoptic area, induced a LH surge in ovariectomised ewes (8, 9). The discovery of a new ER, ERb (10–12), in addition to the classical ER, renamed ERa, lead to new perspectives concerning the mechanisms by which oestrogens may be able to modulate reproduction and, more specifically, GnRH secretion (13). Several studies involving in situ hybridisation (14, 15) and immunocytochemistry (16, 17) have shown a large distribu- tion of ERb in the rodent brain. Although the localisation of ERb mRNA has also been reported in the hypothalamus of the luteal phase ewe (18), there are no reports on the distribution of ERb protein in the brain of this species. Accordingly, our first objective was to map immunocyto- chemically the distribution of ERb expressing cells in the ovine hypothalamus. The distribution of ERb mRNA in the sheep (18) and other species (14–17) overlaps with the known distribution of GnRH neurones. Indeed, a substantial population of double- labelled GnRH/ERb neurones has been reported in the rat (16, 19–21). However, it has not been established whether GnRH neurones express ERb in other species. In this context, a preliminary study on the mouse reported that GnRH neurones do not express immunoreactive ERb (22), although it is well established that GnRH neurones do contain ERb mRNA in this species (23, 24). Thus, our second objective was to test the hypothesis that GnRH neurones express ERb in the ewe. Correspondence to: Dr Donal C. Skinner, University of Wyoming, Department of Zoology and Physiology, Biological Science Building Room 428, Laramie, WY 82071-3166, USA (e-mail: dcs@uwyo.edu). Journal of Neuroendocrinology, 2005, Vol. 17, 29–39 doi:10.1111/j.1365-2826.2005.01271.x Ó 2005 Blackwell Publishing Ltd