Obligatory role of hypothalamic neuroestradiol during the estrogen-induced LH surge in female ovariectomized rhesus monkeys Brian P. Kenealya, Kim L. Keena, James P. Garciaa, Lucille K. Kohlenberga, and Ei Terasawaa,b,1 aWisconsin National Primate Research Center, University of Wisconsin–Madison, Madison, WI 53715; and bDepartment of Pediatrics, University of Wisconsin–Madison, Madison, WI 53715 Edited by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved November 21, 2017 (received for review September 12, 2017) Negative and positive feedback effects of ovarian 17β-estradiol positive feedback effects on the release of GnRH, kisspeptin, and (E2) regulating release of gonadotropin releasing hormone (GnRH) LH in OVX female rhesus monkeys. and luteinizing hormone (LH) are pivotal events in female repro- ductive function. While ovarian feedback on hypothalamo–pitui- Results tary function is a well-established concept, the present study Letrozole Attenuates the Estrogen-Induced LH Surge (Experiment 1). shows that neuroestradiol, locally synthesized in the hypothala- The effects of letrozole or vehicle on the E2-induced LH surge mus, is a part of estrogen’s positive feedback loop. In experiment were examined with a protocol schematically shown in Fig. S1A. 1, E2 benzoate-induced LH surges in ovariectomized female mon- All animals received s.c. implantation of an E2 capsule 14 d before keys were severely attenuated by systemic administration of the systemic EB (day 0, the day of EB injection), and the E2 capsule aromatase inhibitor, letrozole. Aromatase is the enzyme responsi- remained throughout the entire experiment. As shown previously ble for synthesis of E2 from androgens. In experiment 2, using (6), E2 capsule implantation in OVX female monkeys suppresses microdialysis, GnRH and kisspeptin surges induced by E2 benzoate LH levels within a week (compare day −14 to day −7, Fig. 1A). were similarly attenuated by infusion of letrozole into the median Starting at day −7, they were injected daily with letrozole or ve- eminence of the hypothalamus. Therefore, neuroestradiol is an hicle through day 5. Before EB injection (days −5, −3, −1, and 0) integral part of the hypothalamic engagement in response to ele- letrozole, not vehicle, treatment caused a slight, but significant, MEDICAL SCIENCES vated circulating E2. Collectively, we will need to modify the con- suppression of baseline LH levels (Fig. 1B). In the vehicle group, cept of estrogen’s positive feedback mechanism. EB injection on day 0 suppressed LH at 6 h (negative feedback phase), followed by stimulation of LH surge starting at 24 h and neuroestradiol | GnRH | kisspeptin | preovulatory surge | aromatase peaking at 30–48 h after EB injection (positive feedback phase, Fig. 1). The elevated LH release continued up to 54 h after EB. In stradiol (E2), predominantly synthesized by the ovaries, feeds contrast, letrozole treatment significantly attenuated the LH surge Eback to the hypothalamus and anterior pituitary to regulate (Fig. 1). Analysis indicated that both area under the curve (AUC) release of gonadotropin releasing hormone (GnRH) and gonado- and LH surge amplitude in the letrozole group had 67% and 73% tropins, respectively. In addition to the ovary, the brain is also a reductions compared with the vehicle group, respectively (Fig. 2 A B source of E2 synthesis and release. This “neuroestradiol” modulates and ). Letrozole treatment did not alter the latency to or dura- various neuronal functions (1). Previously, we have shown that tion of the LH surge (Fig. 2 C and D). Letrozole treatment did not neuroestradiol, synthesized and released in the median eminence of change elevated circulating E2 levels resulting from estrogen the hypothalamus in ovariectomized (OVX) female rhesus mon- capsule implantation or EB injection (Fig. S2). keys, contributes to control pulsatile GnRH release (2). Neuro- Letrozole Infusion into the Median Eminence Attenuates the estradiol action differs from classical ovarian E2 negative feedback EB-Induced GnRH and Kisspeptin Surge (Experiment 2). The attenu- action. Prolonged infusion of E2 benzoate (EB) into the median eminence, mimicking neuroestradiol release, continuously stimu- ation of the AUC and amplitude of the LH surge by letrozole in lates the release of GnRH and kisspeptin in the median eminence and increases circulating luteinizing hormone (LH) (3), unlike the Significance suppressive effects seen when EB is injected systemically (4). Be- cause the sustained elevation of GnRH and LH release induced by The concept that ovarian cycles in female mammals are regulated prolonged EB infusion into the median eminence is reminiscent of by gonadal steroids released from the ovary is well-established the preovulatory surge (3), we hypothesized that release of neuro- dogma. The findings of this study, however, showing that the estradiol in the median eminence is an integral part of preovulatory removal of neuroestradiol synthesis in the median eminence of the GnRH and LH surges. hypothalamus by the aromatase inhibitor, letrozole, greatly at- During the late follicular phase, the preovulatory gonadotro- tenuates estrogen-induced gonadotropin releasing hormone and pin surge is initiated by increased levels of circulating E2 and luteinizing hormone surges in ovariectomized female monkeys, progesterone from the preovulatory follicle. Experimentally, two indicate that neuroestradiol locally synthesized and released in the different E2 replacement strategies are used for inducing GnRH/ hypothalamus is an integral part of the ovary-initiated positive LH surges in OVX nonhuman primates. An E2 capsule is feedback mechanism of estradiol. implanted into OVX females to mimic LH levels during the follicular phase and 2–3 wk later an additional dose of EB is Author contributions: B.P.K. and E.T. designed research; B.P.K., K.L.K., J.P.G., and L.K.K. injected systemically (5, 6), or a single large dose of EB alone is performed research; B.P.K. and K.L.K. analyzed data; and B.P.K. and E.T. wrote the paper. injected systemically in OVX females (7, 8). Both approaches are The authors declare no conflict of interest. sufficient to induce an LH surge, resembling the preovulatory LH This article is a PNAS Direct Submission. surge (7). GnRH and LH surges induced by systemic adminis- Published under the PNAS license. tration of EB in primates have a latency of ∼24 h and last 36–60 h 1To whom correspondence should be addressed. Email: [email protected]. – (9 11). Using these two GnRH/LH surge models, in the present This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. study we investigated the role of neuroestradiol in estrogen’s 1073/pnas.1716097115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1716097115 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 first 5.7 h in Fig. 3 A and C vs. Fig. 3E and Table S1). Similarly, EB induced kisspeptin surges. Kisspeptin levels during the first 5.7 h in the EB-treated group were significantly higher than the oil control group during the corresponding period (Fig. 3 B and D vs. Fig. 3F and Table S1). Letrozole infusion into the median eminence suppressed GnRH (Figs. 3A and 4A and Table S1) and kisspeptin release (Figs. 3B and 4B and Table S1), whereas vehicle infusion had no effect on GnRH (Figs. 3C and 4A and Table S1)orkiss- peptin release (Figs. 3D and 4B and Table S1). Changes in LH levels in blood samples obtained during the course of experiment 2 (Fig. S1B) suggested that EB suppressed LH levels at 15 h (negative feedback phase, Fig. 5), whereas it stimulated LH release at 36 h EB over baseline LH levels (positive feedback phase, Fig. 5) in both the EB-treated vehicle and letro- zole infusion groups. Importantly, LH levels after letrozole infusion in the EB-treated group at 48 h were significantly lower than those in the EB-treated vehicle infusion group at the corresponding time (Fig. 5). In contrast, the oil-injected group had no changes (Fig. 5). Discussion In the present study, we found that letrozole attenuated the EB- induced LH and GnRH surge by approximately two-thirds in OVX female monkeys. In experiment 1, the latency and duration of the LH surges were not affected by letrozole treatment. This indicates that an elevation of circulating E2, presumably from a preovulatory follicle in the ovary, is necessary to trigger GnRH and LH surges. Once the surge starts, it lasts over 24 h. The results further indicate that a sustained increase in E2 in the hypothalamus is required for full GnRH/LH surges: While elevated E2 in general circulation lasts only ∼30 h (4, 6), the amplitude and the area under the curve (total release) of the LH surge were severely attenuated in the presence of letrozole. Despite the removal of the ovarian source of E2, cells in the hypothalamus and anterior pituitary synthesize E2 (13–15). As such, we speculated that suppression of neuroestradiol synthesis in the median eminence with letrozole during the EB- Fig. 1. Letrozole attenuated the EB-induced LH surge in OVX female induced GnRH/LH surge would modify GnRH levels. Indeed, in monkeys. (A) Changes in LH release from two examples in each (vehicle- and letrozole-treated groups shown in yellow and blue, respectively) during the experiment 2, letrozole infusion into the median eminence reduced entire course of the experiment and (B) group data (mean ± SEM) high- release of GnRH and kisspeptin. Collectively, the results of this lighting the EB-induced LH surge are shown. All animals were implanted with capsules containing estradiol (E2) on day −14 and received EB injection on day 0. In both groups, E2 capsule lowered LH levels, similar to those during the early follicular phase. Analysis indicated that EB induced signifi- cant LH elevations over baseline LH levels before EB injection in both vehicle (n = 5)- and letrozole (n = 5)-treated animals (for both P < 0.001) and that the EB-induced LH surge with letrozole was significantly (P < 0.001) smaller than that with vehicle.