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Estrogen exerts important feedback effects upon the biosynthetic and secretory behavior of gonadotropin-releasing hormone (GnRH) neurons to control reproductive functioning. The mechanism of estrogen action upon these neurons is unclear and seems likely to involve the transsynaptic regulation of GnRH neurons. The objective of the present study was to identify the estrogen-receptive neural populations which project to the general vicinity of the GnRH perikarya in the rostral preoptic area and diagonal band of Broca (rPOA/DBB) of the ewe. Intact breeding-season ewes received an injection of the retrograde tracer fluorogold (FG) into the rPOA/DBB, and their hypothalami and brainstems examined for the presence of FG and estrogen receptor alpha (ERalpha) immunocytochemistry. Retrogradely labeled neurons were identified principally within the lateral septum (LS), lamina terminalis, bed nucleus of the stria terminalis, POA, arcuate nucleus (ARN), ventromedial nucleus (VMN) and median eminence. Smaller numbers of FG-immonoreactive cells were found in the caudal brainstem where they resided mostly in the ventrolateral medulla (VLM). Dual-labeled cells exhibiting both FG and ERalpha staining were prominent in the POA, LS and at all rostrocaudal levels of the VMN and ARN. Small numbers of dual-labeled cells were found in the VLM. These observations indicate that a number of distinct ERalpha-expressing neural populations project to the rPOA/DBB where the majority of the GnRH perikarya are found in the ewe. Although it is not possible to determine the direct connectivity of these projections with GnRH neurons, the findings provide an initial neuroanatomical framework through which the transsynaptic actions of estrogen on ovine GnRH neurons may be tested.

Noradrenergic neurons are implicated in the estrogen-dependent neural regulation of luteinizing hormone secretion in a variety of mammalian species. The current study has used immunocytochemical methods to determine whether estrogen receptors (ER) are expressed within the brainstem of the ewe and to establish their relationship to noradrenergic neurons. Using a monoclonal mouse antiserum directed against the N-terminal of ERa, four distinct populations of ER alpha-immunoreactive cells were identified in ovine medulla and pons. The largest population was found in the superficial laminae of the spinal nucleus of the trigeminal nerve, followed by the nucleus tractus solitarius, lateral area postrema, and ventrolateral medulla. Double-labelling immunocytochemistry using antisera directed against the ER alpha and dopamine-beta-hydroxylase revealed that noradrenergic neurons expressing ER immunoreactivity were only found in ventrolateral medulla (A1 cell group) and nucleus tractus solitarius (A2 cell group). No double-labelled cells were identified in the A5, A6, or A7 noradrenergic cell groups. ERs were expressed with a clear rostrocaudal topography within the A1 and A2 populations, with 80-90% of noradrenergic neurons expressing ERA alpha in the caudalmost medulla as compared with less than 5% rostral to the obex. Our findings demonstrate that, as in the rat, the ovine A1 and A2 neurons express ERs in a defined topographical manner, while, dissimilar to the rat, ER alpha is not synthesized by noradrenergic neurons in the other cell groups. These observations indicate that A1 and A2 noradrenergic neurons in the ovine brainstem are likely to be influenced by circulating estrogens and lay the neuroanatomical foundations for investigating the functional role of these cell populations within the gonadotropin-releasing hormone neuron network of the sheep.