No abstract

A major factor responsible for seasonal anestrus in sheep is a striking increase in the ability of estradiol (E) to inhibit pulsatile GnRH and LH secretion. Previous studies suggest that dopaminergic neurons in the A14 and A15 groups of the ovine hypothalamus play a key role in conveying the inhibitory effects of E in anestrous ewes. The present study tested the hypothesis that A14/A15 neurons in anestrous ewes are activated in response to E, and that this activation is specifically related to seasonal changes in E negative feedback. Expression of the immediate early gene products, Fos and the Fos-related antigens (FRAs), was used as a marker of neuronal activation. Ovariectomized anestrous ewes received either blank implants (no E) or 0.5-cm long E implants sc and were killed 6 h later (E+6h) or 7 days later (E+7d and no E groups). During the breeding season, two additional groups of ovariectomized ewes were perfused 7 days after insertion of either blank or E implants. During anestrus, E completely suppressed LH pulses in the E+7d group, but had no effect in the E+6h group. In the E+7d anestrous group, there was also a significant increase in the mean percentage of tyrosine hydroxylase (TH)-positive cells that expressed nuclear Fos/FRAs in A14 and A15 areas compared to that in either the no E or E+6h group. By contrast, during the breeding season, E had no effect on LH pulse frequency, and there were relatively few TH-positive cells in A14 and A15 that coexpressed Fos/FRAs in either the no E or E+7d group. No significant steroidal or seasonal differences in Fos/FRA expression were seen in other hypothalamic dopaminergic cell groups (A12 and A13) or in the preoptic area-anterior hypothalamus or suprachiasmatic nucleus. Furthermore, E did not alter the total number of TH-positive neurons in A14/A15 or other cell groups. There were seasonal differences in the number of TH-positive neurons, with a significantly greater number of cells in the A13 and A15 of breeding season animals compared to anestrous ewes. Thus, E increased Fos/FRA expression in A14/A15 neurons only during anestrus at a time when it also inhibited LH pulse frequency. These findings are consistent with the view that activation of dopaminergic cells in A14 and A15 is a critical link in the chain of events leading to seasonal shifts in sensitivity to E negative feedback in the ewe.

No abstract

GnRH neurons active in the preovulatory LH surge have been identified in several species using the early intermediate gene product, Fos, but the GnRH neurons active during episodic LH secretion remain unknown. In this study, we have used Fos and Fos-related antigens (FRA) to determine whether a subset of GnRH neurons is active when pulsatile LH secretion is acutely stimulated in sheep. In experiment 1, episodic LH secretion was stimulated in five of six ewes by injection of an opioid antagonist to luteal phase ewes. These five ewes had a 6-fold increase in the percentage of GnRH neurons in the medial basal hypothalamus (MBH) expressing Fos/FRA, compared with control ewes that had no LH pulses before death. Fos/FRA expression was not increased in GnRH neurons found in any other area. In experiment 2, episodic LH secretion was induced in rams by introduction of estrous ewes. This treatment increased Fos/FRA expression in MBH GnRH neurons approximately 10-fold compared with control rams. Again, this increase in Fos/FRA expression in GnRH neurons was limited to the MBH. This selective activation of MBH GnRH neurons could reflect the preferential inhibition of these perikarya by endogenous opioid peptides. It also raises the possibility that a subset of GnRH neurons in the MBH may be responsible for episodic GnRH secretion in sheep.

In the ewe, seasonal anestrus results from a change in the hypothalamic responsiveness to estradiol (E2) negative feedback. Considerable evidence has implicated a specific group of dopaminergic neurons (the A15 group) in this seasonally dependent E2 effect, but these neurons do not appear to contain estrogen receptor-alpha (ERalpha). This apparent discrepancy raises the possibility that at least one other neural system is also involved in mediating E2 inhibition. The purpose of this study was to determine whether ERalpha-containing neurons are activated by the negative feedback action of E2 in anestrus. In Exp 1, we examined the effects of E2 on expression of the immediate early gene products, Fos and Fos-related antigens, in ERalpha-positive cells in anestrous ewes. ERalpha and Fos/Fos-related antigens were colocalized using a dual immunofluorescence procedure in sections throughout the hypothalamus from ovariectomized and E2-treated ovariectomized anestrous ewes. A low dose E2 treatment that inhibited LH pulse frequency and induced Fos in A15 dopaminergic neurons in a previous study significantly increased the percentage of ERalpha-containing neurons expressing Fos (17.8% vs. 1.7%) in the medial preoptic area, but not in other hypothalamic areas. In Exp 2, we determined whether there was a seasonal difference in the effect of E2 on Fos/ERalpha colocalization in this region. E2 treatment produced a 3-fold increase in the percentage of ERalpha-positive cells expressing Fos (15.1% vs. 3.4%) in anestrus, but failed to increase ERalpha/Fos colocalization (1.8% vs. 3.5%) during the breeding season. These data raise the possibility that a subset of ERalpha-containing neurons in the medial preoptic area plays a role in the seasonal change in response to E2 negative feedback in the ewe.