Neural systems mediating seasonal breeding in the ewe

Abstract

Seasonal reproduction in ewes is caused by a dramatic increase in response to oestradiol (E(2)) negative feedback during the nonbreeding (anoestrous) season. Considerable evidence supports the hypothesis that A15 dopaminergic neurones in the retrochiasmatic area (RCh) play a key role in these seasonal changes. These A15 neurones are stimulated by E(2) and inhibit gonadotrophin-releasing hormone (GnRH) secretion in anoestrus, but not the breeding season. Because A15 neurones do not contain oestrogen receptors-alpha (ER alpha), it is likely that E(2)-responsive afferents stimulate their activity when circulating E(2) levels increase during anoestrus. Retrograde tract tracing studies identified a limited set of ER alpha-containing afferents primarily found in four areas [ventromedial preoptic area, RCh, ventromedial and arcuate (ARC) nuclei]. Pharmacological and anatomical data are consistent with GABA- and glutamate-containing afferents controlling A15 activity in anoestrus, with E(2) inhibiting GABA and stimulating glutamate release at this time of year. Tract tracing demonstrated that A15 efferents project posteriorly to the median eminence and the ARC, suggesting possible direct actions on GnRH terminals or indirect actions via kisspeptin neurones in the ARC to inhibit GnRH in anoestrus. Identification of this neural circuitry sets the stage for the development of specific hypotheses for morphological or transmitter/receptor expression changes that would account for seasonal breeding in ewes.

Figure 1

Afferents to A15 based on injections of the retrograde tract tracer CTβ. Panels a and b illustrate injection sites. Panel a: location of the three injection sites. In two ewes (776, 1127), tracer deposits completely overlapped the location of A15 dopamine neurones (filled circles), in another (2017) the tracer deposit partially overlapped the A15. Panel b: ICC staining of CTβ in A15 of ewe 776. Scale bars: 1 mm (a) and 0.2 mm (b). Panels c–F: Drawings of representative sections through the POA (c), rostral (d) and middle (e) and caudal (f) arcuate nucleus (ARC) of ewe 776 summarizing the locations of CTβ-labelled cells (black circles) or cells double-labelled with CTβ and ERα (red circles), ipsilateral (left side) and contralateral (right side) to A15 injection site (grey, shaded area). Each dot represents 5–10 cells, scale bar: 1 mm. Vertical bars in Panels c–f depict mean (± SEM, n=3) number of cells containing CTβ (open bars) or CTβ+ERα (red bars) in areas with major projections to the A15 from a subsequent study using dual immunofluoresence. Panels g and h: dual ICC staining for CTβ (brown) and ERα (black) in tissue from the POA (f) and RCh (g). Abbreviations: ac, anterior commissure; fx, fornix; ir, infundibular recess; mt, mammillothalamic tract; OCh, optic chiasm; OVLT, organum vasculosum of the lamina terminalis; VMH, ventromedial hypothalamus; 3V, third ventricle

Figure 2

Afferents to the A15 from the vmPOA based on injections of the anterograde tracer BDA. Panel a: coronal section through the unilateral BDA injection site on the left (asterisk) also stained for ERα (visible on the right side) illustrating that the injection site overlapped with estrogen-responsive neurones in this area. Panel b: Section through the RCh stained for both BDA (red) and TH (green).

Figure 3

a, b: Low power photomicrographs showing the distribution of CTb-labelled cells and BDA-labelled fibres in the arcuate nucleus (ARC) (a) and median eminence (me) (b) of ewe 776 (see Figure 1). Arrow in a indicates the location of fibres from the A15 coursing medially toward the ARC. Arrow in b indicates efferent fibres coursing through the internal zone of the median eminence; in addition, less abundant efferents (e.g., arrowhead) are seen in the external zone. 3v, third ventricle; pt, pars tuberalis of the pituitary. Scale bar = 100 μm. c, d: Confocal images of sections doubled-labelled for BDA (red) and tyrosine hydroxylase (TH) (green). Double-labelled fibres (arrows) are seen in the ARC (c) and the external zone of the median eminence (d). Scale bars in c = 20 μm; d = 10 μm.

Figure 4

Model for neural circuitry implicated in the seasonal changes in response to E₂ negative feedback. Estrogen-responsive (blue) neurones in the vmPOA and RCh send stimulatory afferents to the A15 DA (red) neurones, which inhibit GnRH (brown) neurones either at terminals in the median eminence or via kisspeptin (green) neurones in the middle and caudal ARC. Insert depicts tissue from the A15 stained ICC for DA (red), GABAergic (blue), and glutamatergic (green) neural elements, illustrating close contacts of the latter two neurotransmitters on to the A15 neurons (arrows). E₂ is postulated to inhibit GABA release and stimulate glutamate release at these synapses.