Neonatal agonism of ERβ by DPN, at the three doses examined, produced only moderate disruption of the HPG axis. Physiological impacts included advanced vaginal opening and premature anestrus. A diminished capacity to elicit Fos labeling in GnRH neurons in response to hormone priming was observed, and although it corresponded with lower LH levels, this effect only reached statistical significance at the highest dose. Impacts on the kisspeptin system were minimal. Reduced Kiss-ir fiber density within the AVPV was only observed at the 1 mg/kg dose and no effects on ARC Kiss-ir were observed. Moreover, peripubertal Kiss1 mRNA levels were not significantly affected by DPN in the AVPV or ARC. In contrast, AVPV expression was significantly abrogated by EB or the ERα agonist PPT. These observations are consistent with what we have reported previously using the 1 mg/kg dose of DPN [4
] and support the hypothesis that ERα plays a more substantial role than ERβ in the estrogen-dependent defeminization of hypothalamic steroid positive feedback signaling pathways during the neonatal critical period.
There was a dose dependent effect of neonatal DPN administration on DOV, a hallmark of puberty in the rat. Although all three doses significantly advanced pubertal onset, none resulted in a DOV as early as the animals exposed to EB. Body weight can be a confounding factor in the timing of pubertal onset, with heavier animals progressing through puberty earlier than lighter animals. This does not appear to have been the case here because there was no significant effect of exposure on body weight. Body weights were equivalent across the groups, and the heaviest animals were in the vehicle treated control group. The 1 mg/kg dose of DPN (MID DPN) advanced puberty by about four days, a shift that is considerably larger than what we have observed previously [4
]. For the present study we used nearly twice as many animals so this difference may at least be partially attributable to the resulting increased statistical power or to other factors we cannot readily account for, such as time of year or quality of maternal care. The specific mechanisms by which estrogen exposure advances DOV remain unclear. Because exogenous administration of kisspeptin has been shown to advance puberty [63
], one possibility is that early DOV results from the premature stimulation of GnRH neurons by kisspeptin. The present results are not consistent with this idea, however, because Kiss1 expression was not elevated in the PND 24 or PND 33 females that ultimately displayed early DOV. In contrast, AVPV levels were significantly decreased in EB exposed females, an effect more indicative of masculinization.
Neonatal agonism of ERβ also significantly curtailed the capacity to maintain a regular estrous cycle. The effect was dose dependent with animals in the group administered the highest dose generally entering a state of anestrus earlier than animals given the lower two doses. By PND 70, more than half of the females in the MID and LOW DPN groups were still cycling, at a point long after nearly all females neonatally exposed to EB or PPT become anestrus [4
]. The observation that the administration of either PPT or DPN can limit the duration over which the estrous cycle progresses normally is consistent with results obtained in a prior study, using different ER selective agonists [65
]. In that study, agonism of ERβ was found to have a more potent effect on estrous cyclicity than agonism of ERα, a result opposite to what we have observed. This could result from the binding and transcriptional properties of the selective agonists, or the timing of exposure. Collectively, however, it is evident from these findings that premature anestrus can result from neonatal exposure to estrogen agonists.
Even though selective agonism of ERβ was not as effective as EB (or PPT) in defeminizing steroid positive feedback on GnRH, it was not completely inconsequential, as the capacity to induce GnRH and Fos co-labeling was reduced by nearly half at the LOW and MID doses and by nearly two-thirds at the HIGH dose. Correspondingly lower LH secretion was also observed, but only at the highest dose. This dose dependent effect of DPN reveals that the capacity to generate GnRH and Fos co-labeling must be abrogated by at least half before appreciable disruption of gonadotropin secretion occurs. This is not entirely remarkable because there are numerous examples where neural cell loss or dysfunction must be substantial before physiological effects are observed. A notable example is Parkinson's disease, in which upwards of 60 to 70% of dopaminergic nigrostriatal neurons must be lost before motor symptoms manifest [66
Although it is generally accepted that hormonal signals are largely conveyed to GnRH neurons by other hormone sensitive neurons within the hypothalamus, GnRH neurons constitutively express ERβ throughout development [67
]. Therefore, it is at least theoretically possible that the partial impairment of steroid positive feedback on GnRH neurons resulted from the direct activation of ERβ on GnRH neurons themselves. Neonatal DPN exposure did not affect the number of GnRH neurons in the anterior hypothalamus, a finding which is consistent with other studies examining the impact of developmental exposure to estrogens or estrogen-like compounds on GnRH neurons [51
], so this cannot be the mechanism by which GnRH neuronal activation was curtailed. An alternative possibility is that DPN acted on other ERβ expressing neurons within the hypothalamus. It is appealing to predict that DPN acted on AVPV Kiss-ir neurons directly, but this hypothesis may prove difficult to test. In the adult female rat, nearly all Kiss-ir neurons are found to be co-localized with either ERα or (to a lesser degree) ERβ [71
] but it appears that neither Kiss1 mRNA nor Kiss-ir are detectable until approximately two weeks after birth [21
]. It is likely that this neuronal population is present but quiescent during this time, but this remains to be clearly established. Thus, it is plausible that early life exposure alters the sex specific organization and function of this neuronal population. A biomarker for these neurons in early neonatal life would be needed to test this hypothesis. Consistent with this idea, however, we have recently shown that there are marked sex differences in ERα and ERβ mRNA expression in the neonatal rat AVPV [22
] with females having higher levels of ERα, but males having higher ERβ levels than females, on the day of birth. This sex difference could at least partially account for why females appear to be more sensitive to the masculinizing influence of ERα agonists at this age. It is also possible that ERβ-expressing neurons with afferents to AVPV Kiss neurons, rather than Kiss neurons themselves, are mediating the decrease in Kiss-ir and GnRH activity. If this is the case, it remains to be determined what those neurons might be. In addition, selective activation of ERβ might have impacted the expression of ERα in the AVPV, and our subsequent observations, therefore, reflect changes in ERα activity [72
]. It has long been hypothesized that each ER subtype modulates the expression and activity of the other in a region specific manner [73
] and this balance may have been disrupted by DPN administration. Regardless of how or where DPN is stimulating ERβ activity, our data show that the net result is only a marginal decrease in AVPV Kiss-ir and GnRH neuronal activation in the adult female.
The impact of DPN on the organization of Kiss-ir in the female AVPV was dose dependent but, unlike the other endpoints we examined, the dose response curve was u-shaped rather than linear. Maximal reduction was observed at the middle (1 mg/kg) dose. In contrast, the suppression of GnRH activation by DPN was approximately equivalent across all three DPN doses and thus consistent with Kiss1 mRNA levels (Figure ), but not concomitant with the u-shaped dose effect on AVPV Kiss-ir levels. It is not readily clear why this one endpoint, and not the others, produced a u-shaped response curve or what the functional significance of it might be. It may simply be a spurious observation, a possibility consistent with the failure to find an appreciable effect of DPN on AVPV Kiss1 expression in peripubertal females. Notably this nonmonotonic type of dose response curve is not atypical for estrogen or estrogen agonists. For example, a biphasic effect of estradiol on terminal end bud formation in the mammary gland and prostate hyperplasia have been described previously [76
]. A definitive mechanism to explain this phenomenon is lacking, but several hypotheses have been put forth, including the differential regulation of hormone receptors with dose, and the overlap of two distinct mechanisms of action [78
]. For the present study, both hypotheses are plausible. In the MID DPN females, LH levels are lower but not significantly so, indicating that the lower numbers of Kiss-ir fibers may have functional significance. Normal levels in the HIGH DPN females may reflect a method by which the system is attempting to compensate for decreased GnRH sensitivity to steroid hormone stimulation. Further studies incorporating serial sampling of LH are needed to explore these possibilities.
Kiss fiber density and mRNA expression was reduced in the ARC of the EB but not the DPN exposed animals, an effect which was not completely unexpected given that ERβ does not appear to be present in the neonate [22
] or adult female ARC [79
]. Many of these fibers likely originate from the AVPV population [81
]. Collectively, the data presented here suggest that ERβ agonism does not alter the density of this plexus. More perplexing, however, Kiss1 mRNA expression was not affected by neonatal PPT exposure nor the combination of DPN and PPT. The significant effect of EB is consistent with what we have seen previously, but remains somewhat surprising given that neither the number of Kiss neurons nor the quantity of Kiss1 expression in the ARC have been found to be sexually dimorphic in rats [26
]. It remains to be determined why neonatal EB administration has such a profound effect on a population of neurons that do not appear to be sexually dimorphic or what the functional significance of this might be. It also remains to be established why the co-administration of DPN and PPT did not recapitulate the effect of EB. It is possible that EB is acting at the level of the membrane or via a "non-classical" pathway to induce the effect [82
As discussed previously, the doses of DPN chosen for this study were selected to bracket the dose of 1 mg/kg that we and others have used before [4
]. Because DPN has only a 70-fold greater relative binding affinity for ERβ than ERα [32
], at high doses it may also begin to bind and activate ERα. Although the highest dose used for the present studies (2 mg/kg) has been successfully employed by others with no evidence of ERα agonsim [36
], it is possible that agonism of ERα occurred to some degree. Partial agonism of ERα could explain why the HIGH DPN females had earlier puberty and lost their estrous cycle more quickly than those in the other two DPN groups, and why GnRH activation was somewhat lower at this dose compared to the other two. Even if agonism of ERα occurs at this dose, from the totality of the data it is evident that agonism of ERβ alone was insufficient to affect any of the endpoints observed to the same degree as EB.