Previously we have shown that rapid action

Previously, we have shown that rapid action of E2in vitro is mediated through two E2 membrane receptors, GPR30 (GPER1) and the STX-sensitive membrane receptors (Noel et al., 2008; Kenealy et al., 2011a, Kenealy et al., 2011b). This is significantly different from receptors implicated in the positive and negative feedback effects of E2. The feedback regulation of E2 requires BW 723C86 hydrochloride receptor ERα (ESR1) or ERβ (ESR2), through which E2 activates nuclear transcription with a longer latency (see Herbison, 2015). As discussed in the following sections, regulation of GnRH neurosecretory activity by neuroestradiol occurs as a rapid E2 action.
Role of neuroestradiol in pulsatile GnRH release During the course of the experiments examining the rapid action of E2 on GnRH release in vivo, we assessed the pattern of local E2 levels as a consequence of EB injection. Results indicate that E2 release in response to EB challenge, measured by liquid chromatography followed by tandem mass-spectrometry (LC/MS/MS), is pulsatile (Fig. 3). The pulsatile release pattern of E2 was rather surprising to us, as we were used to seeing an asymmetric bell shape pattern in circulating E2 after systemic injection of EB, i.e., E2 rapidly increases reaching a plateau, and then slowly returns to the pre-injection level, because the E2 radioimmunoassay is cross-reacting with EB. We soon realized that LC/MS/MS distinguishes E2 from EB because of its different mass-to-charge ratios, such that infusion of EB into the median eminence must have induced local release of E2. If the hypothalamus releases E2 in response to an EB infusion, we should also be able to observe the release without EB treatment. Indeed, we found that electrical stimulation of the medial basal hypothalamus (MBH) of OVX monkeys initially suppresses and then stimulates E2 release (Kenealy et al., 2013). In addition, if the EB-induced E2 release is of hypothalamic origin, we should observe that inhibition of local E2 synthesis blocks stimulatory EB action. Therefore, we examined the effects of the aromatase inhibitor, letrozole, on the EB-induced GnRH and E2 release. In this experiment, we infused letrozole (or its vehicle for control) into the median eminence for 100 min, during which EB was also infused for 20 min. The results indicated that in the presence of letrozole EB did not induce E2 release nor GnRH release, whereas EB induced both GnRH and E2 increases in the absence of letrozole (Fig. 4, Kenealy et al., 2013). Importantly, letrozole also suppressed the EB-induced GnRH release as well as spontaneous GnRH pulses (Fig. 4). At this point we could have concluded that neuroestradiol plays an important role in regulation of pulsatile GnRH release. However, during the course of the study we found that spontaneous neuroestradiol release in the median eminence of OVX monkeys is rare and that when spontaneous release occurs, the amplitude of peaks is much smaller than for peaks evoked by EB or electrical stimulation (Kenealy et al., 2013). Therefore, we concluded that the role of neuroestradiol on regulation of pulsatile GnRH release might be limited to certain circumstances and there must be another role of hypothalamic neuroestradiol in GnRH regulation. I will address this issue with the next series of experiments.
Role of neuroestradiol in GnRH surge A rise in ovarian E2 from the primary follicles is the signal triggering the preovulatory GnRH/LH surge. Using an experimental model, blastula has been shown that systemic injections of EB to OVX monkeys induce GnRH and LH surges, i.e., sustained GnRH and LH elevations for over 36 h with a latency of ~24 h (Levine et al., 1985; Xia et al., 1992; Yamaji et al., 1971; Terasawa, 1985; Schultz and Terasawa, 1988). Because our observations show that prolonged infusion of EB into the median eminence keeps GnRH release elevated for several hours (Kenealy et al., 2015) and that the sustained elevation of GnRH release is reminiscent of the preovulatory GnRH surge, we hypothesized that neuroestradiol release is necessary for the estrogen-induced LH/GnRH surge.