We demonstrated previously that equimolar concentrations of free E2 or E2 conjugated to BSA suppressed the GnRH-induced release of LH in cultures of ovine pituitary cells  suggesting a direct action on the pituitary gland. In OVX ewes, a similar decrease in LH secretion was recapitulated by administration of E2 or E2BSA at equimolar concentrations . In this study we tested the possibility that the decrease in LH secretion induced by E2BSA could (at least partially) be mediated by an action at the level of the hypothalamus. We found that the decrease in LH pulse frequency and mean LH induced by E2BSA was not accompanied by changes in either pulse frequency or pulse amplitude of GnRH. Therefore, the actions of E2BSA on LH secretion seem to occur only at the level of the pituitary gland, presumably by decreasing responsiveness to GnRH. Together with our previous reports [13, 30], these data suggest that pharmacological targeting of the pituitary gland with E2BSA is sufficient to mimic the rapid inhibitory action of E2 on LH secretion. It is worth mentioning that we did not detect a decrease in the pulse amplitude of LH. It is possible that our criterion to detect pulses did not identify small increases in LH concentrations as pulses, or that our sampling was too infrequent to detect a marginal decrease in pulse amplitude. The exclusion of these "low amplitude pulses" may have masked an effect on the amplitude of LH pulses. Nevertheless, since E2BSA did not decrease either pulse frequency or pulse amplitude of GnRH, the effect of E2BSA on LH secretion is attributed to a direct action on the pituitary gland. The apparent lack of effect of E2BSA on the secretory pattern of GnRH provides evidence that E2BSA does not have a hypothalamic effect, possibly because it does not cross the blood-brain barrier. In a recent study  evidence was provided for the mechanism underlying the rapid inhibitory effect of E2 on the GnRH-induced release of LH secretion in primary cultures of ovine pituitary cells. In that study, the increase in cytoplasmic intracellular free calcium concentration ([Ca++]i) mediating the GnRH-induced release of LH secretion was abolished by 2 min pretreatment with E2 or E2BSA, which agrees with the time frame for the beginning of the rapid decrease of LH secretion induced by estrogens. In hypothalamus-pituitary-disconnected ewes, E2 increased the number of gonadotropes expressing phosphorylated extracellular signal-regulated-kinases 1 and 2 (pERK-1/2), c-AMP-responsive element-binding protein (pCREB), and serine 473 kinase (pAkt) within 15 to 90 minutes after administration of E2 . Whether these and/or other upstream signaling molecules contribute to maintain a decreased responsiveness of the pituitary gland to GnRH (for at least 10 h in our previous study ) after administration of estrogens is yet to be evaluated.
A caveat of using E2BSA as a tool to study non-genomic actions is the stability of the conjugate, particularly if the conjugate is used in studies in vivo. We demonstrated  that conjugated forms of E2 mimicked the acute non-genomic action of E2 (i.e. rapid decrease of LH secretion), but not the more prolonged, presumably genomic actions (i.e. preovulatory-like surge of LH and decrease in FSH secretion). These data strongly support the conclusion that there is insufficient free E2 (i.e. enough to mimic the genomic actions) in the conjugate to have a biological effect. In the current study we provided further evidence on the stability of E2BSA in blood. It is known that E2 can rapidly regulate secretion of GnRH [8, 16, 18, 43, 44]. Consequently, the fact that E2BSA did not change the secretory pattern of GnRH strengthens our previous conclusion of the absence of sufficient free E2 to induce a physiological response. Furthermore, administration of E2BSA to OVX ewes did not raise serum concentrations of free E2 over pre-treatment levels during the 24 hours evaluated. Therefore, the actions of E2BSA can only be attributed to the conjugated form of E2.
In a previous report , we showed that infusion of E2BSA to OVX ewes did not induce a typical preovulatory-like surge of LH; instead a slight but significant increase in serum concentrations of LH was detected at the time of the expected massive release of LH. The possibility that the slight increase in LH secretion induced by E2BSA was the result of an increase in the number of GnRH receptors was evaluated in this study. Indeed, administration of E2BSA to OVX ewes increased the number of GnRH receptors in the pituitary gland and it was associated with a slight rise in serum concentrations of LH at the expected time for the beginning of the preovulatory-like surge of LH (12 h after administration of conjugated E2). This report provides the first in vivo evidence in ewes of a membrane-initiated pathway as a component of the mechanisms underlying the synthesis of GnRH receptors induced by E2. It seems that the non-genomic actions of E2 in the pituitary gland are not restricted to the rapid suppression of pulses of LH reported elsewhere . The ability of E2BSA to induce both a suppression in responsiveness of the pituitary gland to GnRH and an increase in the number of GnRH receptors is compatible since E2 up-regulates the number of GnRH receptors subsequent to the short-term negative feedback [2, 6, 45, 46] and the sum of these two events may be part of the strategy to ensure a buildup of pituitary content of LH for a robust preovulatory surge. Since an ERE is not involved in mediating the actions of E2BSA [24, 27, 47, 48] and a canonical ERE has not been reported in the GnRH receptor gene [47–50], we speculate that a membrane-initiated pathway may activate down-stream events facilitating the transcription of GnRH receptors, as well as inhibiting GnRH-induced secretion of LH. As mentioned above, an increase in phosphorylation level of a number of proteins in the gonadotropes has been reported 15 to 90 minutes after administration of E2 . The ability of E2 or E2BSA to increase the level of protein phosphorylation for longer periods would be compatible with our speculation on a membrane-initiated pathway as a component in the synthesis of GnRH receptors number, as well as maintaining the inhibition of LH secretion after the initial decrease. The ability of E2 and E2BSA to activate upstream signaling molecules for longer intervals has yet to be evaluated.
The ability of E2BSA to inhibit responsiveness of primary cultures of ovine pituitary cells to GnRH  was the basis for examining its ability to abolish the E2-induced massive release of LH in ewes. Based on our previous in vivo study  E2BSA would decrease responsiveness of the pituitary gland to GnRH within the first hour after E2BSA, and it remained decreased for at least 10 h under basal secretion of GnRH. In the current experiment we overlap the period of a decreased responsiveness of the pituitary gland to GnRH to the expected time for the surge of LH by administering E2BSA 8 h after E2 (2-3 h before the beginning of the preovulatory-like surge of LH) at a dose 10 times higher than previously reported . Under these experimental conditions, E2BSA failed to obliterate completely the E2-induced massive release of LH. Nevertheless, E2BSA decreased the magnitude of the preovulatory surge of LH induced by E2, not only delaying the onset of the surge of LH, but also resulting in an early termination of the preovulatory-like surge of LH, and to some extent reducing the amount of LH released. It is not clear why the decrease in the amount of LH released by E2BSA was more apparent in ewes highly responsive to E2 than in ewes with a lower responsiveness to E2. If as proposed, E2BSA is reducing responsiveness of the pituitary gland to GnRH, it is more likely that E2BSA could override the GnRH input during the beginning and the end of the surge of LH, when secretion of GnRH is not at the maximum concentration [1, 4, 8, 16, 17, 19]. Notwithstanding the expected increase in the number of GnRH receptors, the net effect of E2BSA was a reduction in the magnitude of the preovulatory-like surge of LH. This implies a predominant role of the membrane-initiated actions of E2 in the mediation of the negative feedback effect on LH secretion compared with a minor contribution, by itself, on the synthesis of GnRH receptor. This interpretation is consistent with the slight increase in the number of GnRH receptors induced by E2BSA, compared with that induced by E2, and agrees with other experimental paradigms where equimolar doses of conjugated or un-conjugated E2 induced a similar decrease in LH secretion in ewes,  and in primary cultures of ovine pituitary cells ; whereas in the same cells, higher doses of E2BSA were required to increase the number of GnRH receptors .
Although regulation of synthesis and/or secretion of GnRH by E2BSA were not evaluated during the expected time of the preovulatory-like surge of LH, we do not think that the effects were mediated via the hypothalamus. In support of this notion: a) E2BSA did not modulate the pulsatile profile of GnRH (at least for the first 4 h after administration), b) conjugated E2 was stable in blood (at least for 24 h), and c) administration of a high dose of E2BSA just before the preovulatory-like surge of LH induced by E2 decreased the amount of LH released. Therefore we suggest that the actions of E2BSA on LH secretion are the result of a direct action on the pituitary gland.